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The spatial separation of processing and transport functions to the interior and periphery of the Golgi stack

  1. Hieng Chiong Tie
  2. Alexander Ludwig
  3. Sara Sandin
  4. Lei Lu  Is a corresponding author
  1. Nanyang Technological University, Singapore
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Cite this article as: eLife 2018;7:e41301 doi: 10.7554/eLife.41301

Abstract

It is unclear how the two principal functions of the Golgi complex, processing and transport, are spatially organized. Studying such spatial organization by optical imaging is challenging, partially due to the dense packing of stochastically oriented Golgi stacks. Using super-resolution microscopy and markers such as Giantin, we developed a method to identify en face and side views of individual nocodazole-induced Golgi mini-stacks. Our imaging uncovered that Golgi enzymes preferentially localize to the cisternal interior, appearing as a central disk or inner-ring, whereas components of the trafficking machinery reside at the periphery of the stack, including the cisternal rim. Interestingly, conventional secretory cargos appeared at the cisternal interior during their intra-Golgi trafficking and transiently localized to the cisternal rim before exiting the Golgi. In contrast, bulky cargos were found only at the rim. Our study therefore directly demonstrates the spatial separation of processing and transport functions within the Golgi complex.

https://doi.org/10.7554/eLife.41301.001

Introduction

The Golgi complex is one of the most important processing and sorting stations along the secretory and endocytic pathway (Glick and Luini, 2011; Klumperman, 2011; Lu and Hong, 2014). In mammalian cells, it consists of a network of laterally linked Golgi stacks. As the structural unit, a Golgi stack comprises 4–7 flattened cisternae and can be divided into cis, medial and trans-regions. The trans-Golgi region further develops into the trans-Golgi network (TGN). It is known that the cis-Golgi receives secretory cargos from the endoplasmic reticulum (ER) exit site (ERES) and ER Golgi intermediate compartment (ERGIC), while the trans-Golgi and TGN exchange materials with endosomes and the plasma membrane (PM). At the moment, we still don’t understand how the Golgi becomes organized and works at the molecular and cellular level (Glick and Luini, 2011). One of the challenges in studying the Golgi is to spatiotemporally resolve residents and transiting cargos among individual cisternae of Golgi stacks, a task currently beyond the capabilities of even super-resolution and electron microscopy (EM).

It has been hypothesized that the two principal functions of the Golgi, processing and transport, are spatially organized for optimal efficiency (Patterson et al., 2008). However, such molecular organization across the Golgi stack has not been directly demonstrated. Previously, by utilizing nocodazole-induced Golgi mini-stacks, we developed a conventional microscopy based super-resolution method, named GLIM (Golgi localization by imaging center of fluorescence mass), to quantitatively map the axial position or localization quotient (LQ) of a Golgi protein with nanometer accuracy (Tie et al., 2017; Tie et al., 2016b). To understand the molecular organization of the Golgi mini-stack, the lateral localization, which refers to the distribution of a protein within Golgi cisternal membrane sheets, is also required. Although more structural details of the Golgi can be resolved with the advent of the super-resolution microscopy, it is still difficult to unambiguously interpret Golgi features due to the dense packing of stochastically oriented Golgi stacks. Here, we established a method to systematically study the lateral localization of Golgi proteins. We found that Golgi enzymes and components of trafficking machinery are spatially separated to the interior and periphery, respectively, of the Golgi stack, while secretory cargos with bulky sizes are excluded from the interior during their intra-Golgi transition.

Results

Giantin, GPP130 and Golgin84 localize to the cisternal rim of the Golgi mini-stack

There have been extensive evidences demonstrating that the nocodazole-induced Golgi mini-stack is a valid model of the native Golgi (Cole et al., 1996; Rogalski et al., 1984; Trucco et al., 2004; Van De Moortele et al., 1993) and we have previously discussed its advantages in studying the molecular and spatial organization of the Golgi (Tie et al., 2017; Tie et al., 2016b). Apparently, the lateral localization of a Golgi protein is best revealed by its en face and side view, when the Golgi axis is roughly orthogonal and parallel, respectively, to the image plane. We found that the orientation of a mini-stack can be identified by Golgi markers, such as Giantin, Golgin84 and GPP130. Airyscan super-resolution microscopy clearly revealed their staining patterns as rings (Figure 1A). Assuming cisternae of a Golgi mini-stack are round membrane disks, we reasoned that these proteins must localize to the rim of their corresponding cisternae and their ring appearances must correspond to en face or oblique views (hereafter en face views) (Figure 1B). As expected for the orthogonal section of a ring (Figure 1B), side view images of Giantin, Golgin84 and GPP130 displayed a double-punctum, the connecting line of which is roughly orthogonal to the Golgi-axis (Figure 1C). To describe the localization pattern of a population of mini-stacks, we developed a method to average multiple en face view images of Golgi mini-stacks, by applying size and intensity normalization followed by alignment according to their centers of fluorescence mass (see Materials and methods). En face averaged Giantin, Golgin84 and GPP130 demonstrated their lateral localization patterns as concentric circular rings of difference sizes (Figure 1D–F). To substantiate our light microscopic data, we imaged APEX2-fused GPP130 by EM using native NRK cells that were not subjected to nocodazole treatment (Figure 1G; Figure 1—figure supplement 1A,B). Out of 57 Golgi stacks that we randomly imaged from 25 cells, 68% demonstrated a predominant cisternal rim localization in side or en face views (Figure 1—figure supplement 1C), supporting the ring staining pattern observed. The rim localization of Giantin was also corroborated in a previous immuno-EM study, furthring supporting our data (Koreishi et al., 2013).

Figure 1 with 2 supplements see all
Identifying the en face and side view of the Golgi mini-stack.

All cells are nocodazole-treated HeLa cells and all images are super-resolution images unless specified otherwise. By default, tagged-proteins were transiently transfected while non-tagged proteins were native and stained by their antibodies. (A) The staining patterns of Giantin, Golgin84 and GPP130 appear as concentric rings. (B) The schematic representation of different orientation views (en face, oblique and side) of a Golgi cisterna and the corresponding expected images of a rim-localized protein (colored as pink). (C) The double-punctum appearances of Giantin, Golgin84 and GPP130 indicate side views of Golgi mini-stacks. In each merge, the intensity profile is generated along a thick line, represented by a dotted box, with the direction indicated by the arrow (the same scheme is used throughout this study). The dotted box schematically marked the start, end and width of the line. The direction arrow roughly follows the cis-to-trans Golgi axis using the cis-most (GM130 in this case) and trans-most markers in each panel. Dotted pink lines connecting double-punctum are almost orthogonal to the cis-to-trans Golgi axis. The intensity plot is normalized and color-coded as the corresponding merge image. (D–F) En face averaged images of Giantin, fluorescence protein (FP)-Golgin84 and GPP130-GFP. The corresponding radial mean intensity profile is shown at the right with distance from the center of fluorescence mass (normalized to the radius of Giantin) as the x-axis and radial mean intensity (normalized) as the y-axis. Both GFP and mCherry-tagged Golgin84 images were used for FP-Golgin84. n, the number of averaged Golgi mini-stacks. (G) GPP130 mostly localizes to the cisternal rim (arrows) of the native Golgi by EM. NRK cells transiently expressing GPP130-APEX2-GFP were subjected to APEX2-catalyzed reaction followed by EM. Note that cells were not subjected to nocodazole treatment. The EM thin section image displays the side view of a Golgi mini-stack. The electron density indicates the localization of GPP130 (arrows). (H) The histogram showing the distribution of diameters of Giantin-rings. (I, J) Giantin N and C-terminus colocalize at the cisternal rim. In (I), cells were co-stained using Giantin antibodies raised against its N and C-terminus. In (J), Giantin N-terminus was stained by an antibody and its C-terminus was revealed by exogenously expressed mScarlet-Giantin-C129. In the en face view, dotted arrow represents the line used to generate the line intensity profile (width = 1 pixel), while in the side view, the dotted box that is in the direction of the arrow and parallel to the Golgi cisterna represents the line for intensity profile. (K) The interior localization of MGAT2 within the Giantin-ring. Line intensity profiles of the en face and side views are acquired as those in (I) and (C) respectively. Scale bar, 500 nm.

https://doi.org/10.7554/eLife.41301.002

Among all Golgi markers, we observed that Giantin had the largest ring diameters—950 ± 10 nm (mean ± SEM, same for the rest; n = 336) (Figure 1H). It is known that the epitope of our antibody is at the N-terminus while Giantin anchors onto the Golgi membrane via its extreme C-terminal transmembrane domain (Linstedt et al., 1995). A fully extended Giantin molecule is predicted to reach 450 nm (Munro, 2011). Hence, it is possible that the large diameter of Giantin-ring can be due to Giantin’s extended structure instead of the physical dimension of Giantin-positive cisternae. However, we think this is not the case due to our following observations. First, we raised an antibody against its C-terminal cytosolic region and ring-patterns resulted from N- and C-terminal antibodies colocalized very well (Figure 1I). Quantitative analysis revealed that the mean diameter of the C-terminus ring is ~50 nm smaller than that of the N-terminus one (Figure 1H; Figure 1—figure supplement 2A), far less than the value predicted for the fully extended molecule, which is 900 nm. Second, the C-terminal 129 amino acid fragment of Giantin (mScarlet-Giantin-C129), which has a LQ similar to native Giantin (Table 1), displayed almost the same ring-pattern as the N-terminal antibody (hereafter Giantin antibody unless indicated otherwise) (Figure 1J). Third, similarly, the N- and C-termini of other Golgins such as GM130 and GCC185 also showed overlapping ring-patterns (Figure 1—figure supplement 2B,C). In summary, although individual Golgins might adopt long filamentous conformation (Munro, 2011), ensemble-averaged Golgins, as visualized in bulk by light microscopy, appear to have a closely adjacent N- and C-termini (Cheung et al., 2015). Therefore, the ring-pattern staining of Giantin should closely represent the cisternal rim.

Table 1
List of LQs used in this study.

Please see Table 1-table supplement 1 for official full names of glycosylation enzymes.

https://doi.org/10.7554/eLife.41301.005
NameLQNSEM
Myc-Sec13−0.96390.09
β-COP$−0.70740.11
Arf4-GFP−0.61510.07
Sec23a-mCherry−0.581210.06
Arf5-GFP−0.46420.06
GS27*,$−0.221010.03
γ-COP$−0.171060.07
GFP-ERGIC53*−0.161980.02
KDEL receptor*, $−0.111300.03
GFP-GM130*−0.05930.04
GM130*, $, #0.00--
GRASP65-GFP0.021980.01
GRASP55-GFP0.071400.02
GFP-Rab1a0.211540.03
ManII-SBP-GFP0.23530.05
GFP-ACBD30.251320.03
GFP-Golgin84*0.261080.03
Man1B1-Myc0.42880.05
β3GalT6-Myc0.47970.03
MGAT4B-AcGFP10.50230.04
β4GalT7-Myc0.521100.04
MGAT2-Myc0.531360.04
GS28*0.531250.08
MGAT2-AcGFP10.561100.04
Giantin$0.571030.05
TPST2-GFP*0.641540.02
POMGNT1-Myc0.67870.04
MGAT1-Myc0.701410.02
GPP130-APEX2-GFP0.711000.03
Myc-Sec340.71270.12
β4GalT3-Myc0.741490.02
Arf1-GFP0.75870.03
TPST1-GFP*0.761110.04
ST6Gal1-Myc0.761540.03
mScarlet-Giantin-C1290.801610.01
GS15$0.831500.03
GPP130-GFP*0.841680.02
SLC35C1-Myc0.84850.04
ST6Gal1-AcGFP10.851380.02
GALNT2$0.861070.03
GFP-GCC1850.941220.05
GALNT1$0.97900.02
GalT-mCherry*,#1.00--
GFP-Rab6*1.042620.04
Arl1*, $1.20260.05
Vti1a*, $1.261620.02
GFP-GGA11.30330.12
Golgin245*, $1.421260.05
GFP-Golgin97*1.451610.03
CI-M6PR*, $1.46420.24
Syntaxin6*, $1.56840.11
Vamp4-GFP*1.571570.04
Furin*, $1.62430.11
CLCB$1.65370.26
GGA2*, $1.96330.23
  1. *,previously published data (Tie et al., 2016b);

    $, endogenous protein.

  2. #, LQs of GM130 and GalT-mCherry are defined as 0.00 and 1.00 (Tie et al., 2016b).

Identifying en face and side views of Golgi mini-stacks

By assessing the super-resolution staining patterns of Giantin, GPP130 or Golgin84, we can conveniently identify en face and side view oriented Golgi mini-stacks, images of which should appear as a ring and double-punctum, respectively. It was discovered that some Golgi residents, such as MGAT2, localized to the interior of Giantin-rings (Figure 1K). Consistent with this interpretation, side views of MGAT2 appeared as a short bar connecting the Giantin double-punctum (Figure 1K). Under the EM, MGAT2-APEX2-GFP preferentially localized to the cisternal interior (next section). Therefore, there are at least two types of lateral localizations: rim and interior, as represented by Giantin and MGAT2.

Golgi trafficking components mainly localize to the periphery of a Golgi mini-stack

We systematically examined the lateral localization of Golgi residents using their en face and side views. Two types of residents were studied in this work — components of trafficking machinery, including those involved in the structure and organization of the Golgi, and enzymes involved in the post-translational modifications, particularly glycosyltransferases. Due to the lack of reagents to detect endogenous proteins, many residents were detected by the overexpression of their tagged fusions (Table 1). Caution must be taken in the interpretation of our data as it has been documented that overexpression can change both the axial and lateral localization of Golgi residents (Cosson et al., 2005). We discovered that the lateral localization of trafficking machinery components shares common features according to their LQs.

ERES, ERGIC and cis-Golgi proteins (LQ <0)

COPII coat subunits, including Sec13 and Sec23a, COPI coat subunits, including β and γ-COP, KDEL receptor, GS27, ERGIC53, Arf4 and Arf5, displayed lumps or puncta around Giantin-rings in en face views and at one side of Giantin-double-punctum in side views (Figure 2A–D; Figure 2—figure supplement 1A–E).

Figure 2 with 1 supplement see all
Components of the ERES, ERGIC and cis-Golgi transport machinery mainly localize to the periphery of the Golgi mini-stack.

(A–D, E and H) Typical en face and side view images of Golgi transport machinery components. (A–D) ERES, ERGIC and cis-Golgi proteins (LQ <0). (E and H) cis-Golgi proteins (0 ≤ LQ < 0.25). (F–I) En face averaged images and radial mean intensity profiles corresponding to (E) and (H). n, the number of averaged Golgi mini-stack images. Scale bar, 500 nm.

https://doi.org/10.7554/eLife.41301.006

cis-Golgi proteins (0 ≤ LQ < 0.25)

GM130, GRASP55, GRASP65 and Rab1a mainly appeared as a central disk and bar in en face and side views, respectively (Figure 2E–I; Figure 2—figure supplement 1F,G). When they appeared as rings in en face views, there were usually some interior tubular or sheet connections (Figure 2E,H; Figure 2—figure supplement 1F). Both observations suggest that these proteins probably localize throughout cis-cisternae.

Medial and trans-Golgi proteins (0.25 ≤ LQ < 1.0)

ACBD3, Golgin84, Giantin, GS15, GS28, Sec34, GPP130 and GCC185, all displayed ring-pattern localizations (Figure 1A,C; Figure 3A–F;Figure 3—figure supplement 1A–D), suggesting that they mainly localize to the rim of their corresponding cisternae and are mostly absent from the cisternal interior. Arf1, whose LQ is 0.75, is an exception here. Although its en face view demonstrated that it is in the cisternal interior, side view images uncovered that there were two pools: a cis/medial and a trans-Golgi/TGN pool, with a much reduced presence in between (Figure 3G,H). This observation is consistent with the notion that Arf1 functions in the cis-Golgi and TGN for the assembly of the COPI and clathrin coat, respectively (Gillingham and Munro, 2007).

Figure 3 with 2 supplements see all
Components of the medial, trans-Golgi and TGN transport machinery mainly localize to the periphery of the Golgi mini-stack.

(A–H) Medial and trans-Golgi proteins (0.25 ≤ LQ < 1.0), except Arf1, localize to the cisternal rim. En face and side view images are shown. Corresponding en face averaged images and radial mean intensity profiles are shown in (B, D, F and H). n, the number of averaged Golgi mini-stack images. (I–L) trans-Golgi and TGN proteins (LQ ≥1.0) appear compact or scattered at one end of the mini-stack. Arrows in (I) indicate colocalization between CLCB and Vamp4-GFP. Scale bar, 500 nm.

https://doi.org/10.7554/eLife.41301.008

trans-Golgi and TGN proteins (LQ ≥1.0)

There are two types of localization patterns at the trans-side of Giantin-rings. The distribution of Vamp4, Golgin97, Vti1a, Syntaxin6, Rab6, Arl1 and Golgin245 was relatively compact (Figure 3I,J; Figure 3—figure supplement 2A–G). In contrast, GGA1, GGA2, clathrin light chain B (CLCB), CI-M6PR and Furin showed punctate or tubular profiles (Figure 3I,K,L; Figure 3—figure supplement 2H–L). Although all are TGN proteins, most of them did not exhibit appreciable colocalization. For example, Vamp4 did not show a significant overlap with CI-M6PR, GGA2, Furin, or Vti1a (Figure 3L; Figure 3—figure supplement 2K–M). However, CLCB was found to decorate punctate and tubular profiles of both Vamp4 (Figure 3I) and Furin (Figure 3—figure supplement 2H) outside the stacked cisternal membrane, in agreement with 3D EM-tomography of the TGN (Ladinsky et al., 1999) and the role of clathrin coat in transporting these cargos to the endolysosome (Peden et al., 2001; Teuchert et al., 1999). Our data are also consistent with the notion that the TGN comprises domains of distinct molecular compositions (Brown et al., 2011; Derby et al., 2004).

In summary, our extensive super-resolution imaging data suggest that Golgi trafficking components mainly localize to the entire cis-cisternae, rim of medial and trans-cisternae and punctate or tubular profiles at non-stacked regions, which include the ERES, ERGIC and TGN.

Glycosylation enzymes reside at the interior of a Golgi stack

We studied components of Golgi post-translational modification machinery (Table 1; Supplementary file 1), including a GDP-fucose transporter, SLC35C1 (Lübke et al., 2001), and more than a dozen enzymes involved in N-glycosylation (Man1B1, MGAT1, ManII, MGAT2, GalT, SialT and MGAT4B), O-glycosylation (GALNT1, GALNT2 and POMGNT1), poly-N-acetyllactosamine synthesis (β4GalT3), glycosaminoglycan synthesis (β3GalT6 and β4GalT7) and sulfation (TPST1 and 2). Interestingly, their LQs were found to be in the range from 0.23 to 1.0 (Table 1), suggesting that Golgi enzymes mainly localize to the medial and trans-region of the Golgi, but not to the cis-Golgi and TGN. This observation is consistent with previous EM studies. For example, in plant cells, polysaccharides were mainly detected in the medial and trans-Golgi cisternae (Zhang and Staehelin, 1992). Similarly, in mammalian cells, the N-glycan modifying enzymes ManI, ManII and MGAT1 have been mapped to the medial and trans-region of the Golgi stack (Dunphy et al., 1985; Nilsson et al., 1993; Rabouille et al., 1995; Velasco et al., 1993). However, in contrast to our quantitative results, previous EM work has assigned GalT (Nilsson et al., 1993; Rabouille et al., 1995; Roth and Berger, 1982) and SialT (Rabouille et al., 1995; Roth et al., 1985) to the TGN in addition to the trans-Golgi. Sub-Golgi localizations are not always consistently reported, which is likely due to two reasons. First, the cis, medial, trans-region and TGN are not rigorously defined and the assignment of Golgi regions can be subjective. Second, it has been documented that the sub-Golgi localization of enzymes can be cell-type dependent (Velasco et al., 1993).

In contrast to trafficking components, our Golgi enzymes and SLC35C1 localized within Giantin-rings as a central disk in en face views (Figure 1K; Figure 4A–D; Figure 4—figure supplement 1A–T), except Man1B1, ManII, MGAT4B and TPST2, which mostly appear as an inner-ring concentric to the corresponding Giantin-ring (Figure 4E,F; Figure 4—figure supplement 2A–F). The disk and ring patterns were more obviously revealed after en face averaging (Figure 4B,D,F; Figure 4—figure supplement 1B,D,F,H,J,L,N,P,R,T; Figure 4—figure supplement 2B,D,F). Since MGAT2-Myc and MGAT4B-AcGFP1 had almost the same LQs as Giantin (mean values: 0.53 and 0.50 vs 0.57 respectively) (Table 1), a significant amount of these proteins are expected to reside in the same cisternae. The lateral distribution pattern of MGAT2 and MGAT4B suggests that they should mainly localize to the interior of cisternae as a central disk and inner-ring, respectively, within the Giantin-rim in the same cisternae (Figure 4G). Enzymes, such as β4GalT3 and ST6Gal1, which have similar LQs (Table 1), were observed to localize to shared and distinct domains within Giantin-rings (Figure 4H).

Figure 4 with 3 supplements see all
Golgi enzymes primarily localize to the interior of medial and trans-Golgi cisternae.

(A, C and E) En face view images of Golgi enzymes. Side view images are also shown in (A) and (C). Dotted arrows and boxes and line intensity profiles are used or acquired as in Figure 1K. (B, D and F) Corresponding en face averaged images and radial mean intensity profiles. n, the number of averaged Golgi mini-stack images. (G) The merge of en face averaged images of Giantin, MGAT4B and MGAT2 and the corresponding radial mean intensity profile. n, the number of averaged Golgi mini-stack images. (H) β4GalT3 and ST6Gal1 can localize to shared (arrows) and distinct domains within the cisternal interior. (I) MGAT2 localizes to the cisternal interior of the native Golgi by EM. NRK cells transiently expressing MGAT2-APEX2-GFP were subjected to APEX2-catalyzed reaction followed by EM. Note that cells were not subjected to nocodazole treatment. The thin section EM image displays the side view of a Golgi stack. MGAT2-APEX2 positive cisternal interior and budding profiles are indicated by arrows and arrow heads, respectively. (J) A quantitative molecular map of the Golgi mini-stack. The normalized radius of a Golgi protein is plotted against its corresponding LQ (Table 1). Red open and closed circle denote ring and disk lateral localization pattern, respectively. n, the number of Golgi mini-stacks used to calculate normalized radius. (K,L) Identifying the rim and interior of native Golgi cisternae. Cells were not treated with nocodazole. In (K), the cisternal rim (arrows) and interior are labeled by Giantin and β4GalT3, respectively. In (L), Giantin and GPP130 positive curvy lines (arrows) represent cisternal rim and do not correspond to side views or cross sections of Golgi stacks. The boxed region in each image is enlarged in the upper right corner. Scale bars represent 500 nm unless specified otherwise.

https://doi.org/10.7554/eLife.41301.011

To substantiate our light microscopic data, we examined the localization of MGAT2-APEX2-GFP in the native Golgi by EM. 93% (n = 58) of Golgi stacks showed an enrichment of MGAT2 in the cisternal interior (Figure 4I; Figure 4—figure supplement 3A–C), which is in contrast to the staining pattern observed for GPP130 (Figure 1G). Noticeably, APEX2-generated electron density was also found in vesicles and budding profiles at the rim (arrow heads in Figure 4I). However, we did not find MGAT2-AcGFP1 (Figures 1K and 4C) or MGAT2-APEX2-GFP (Figure 4—figure supplement 1U) signal outside Giantin-rings by fluorescence imaging of Golgi mini-stacks. Although the identity and destiny of these vesicles are currently unknown, our observations suggest that Golgi enzymes might be depleted from the rim either by retrieval to the interior or by sorting into membrane carriers. Together, our data demonstrate that Golgi enzymes mainly localize to the interior of medial and trans-cisternae as a concentric disk or inner-ring, while trafficking machinery components exhibit rim localization.

A quantitative molecular map of the Golgi mini-stack

To quantitatively describe the overall lateral distribution of Golgi proteins, we assume that a Golgi protein has a radial symmetry localization around the Golgi axis as a concentric disk or ring. The normalized radius of the ring or disk can be measured using the radial mean intensity profile of en face averaged images (see Materials and methods). A plot of the normalized radius versus LQ quantitatively summarizes our morphological observations of ring and disk distribution of various Golgi residents (Figure 4J). While medial and trans-Golgi trafficking machinery components are at the cisternal rim, Golgi enzymes all localize to the interior with Man1B1, ManII, MGAT4B and TPST2 appearing as concentric inner-rings and the rest as central disks. Interestingly, it also reveals that cis-cisternae have smaller diameters than medial ones, consistent with many EM thin-section or tomographic 3D images (Bykov et al., 2017; Engel et al., 2015; Staehelin and Kang, 2008), though the biological significance of which remains to be further investigated.

Imaging the organization of the native Golgi complex

Having studied in detail the organization of Golgi mini-stacks, we attempted to resolve the organization of the native Golgi complex by the super-resolution microscopy. Giantin and Golgi enzymes were used to mark the rim and interior of stacked cisternae, respectively. In the less dense region, Giantin and GPP130 staining appeared as distinctive ring- or loop-patterns, with β4GalT3 and GM130 filling the interior (Figure 4K,L), similar to the nocodazole-induced mini-stack. β4GalT3 and GM130 positive membrane sheets likely correspond to stacked Golgi cisternae. In most cases, Giantin and GPP130 positive curvy lines did not correspond to side views or cross-sections of Golgi stacks. Instead, they corresponded to the rim of cisternae in oblique or en face views (arrows in Figure 4L). In the more densely packed region, cisternae appeared to pile on top of each other, a configuration that requires much higher z-axis resolution to be resolved. Nonetheless, we demonstrated that, aided with suitable markers, it is possible to identify the cisternal rim and interior of the native Golgi complex by light microscope.

The lateral localization of secretory cargos during their intra-Golgi trafficking

To study the lateral localization of secretory cargos during their intra-Golgi trafficking, the retention using selective hooks (RUSH) system was adopted to synchronously release secretory cargos (Boncompain et al., 2012). The RUSH reporter CD59, a GPI-anchored protein, was first detected in the interior of cis-Golgi cisternae after 10 min of chase (Figure 5A,B). During its transition through the Golgi mini-stack, as evidenced in its LQ versus time plot (Figure 5B), CD59 remained in the interior (Figure 5A), although its total intensity in Golgi mini-stacks initially increased and subsequently decreased due to the export toward the PM. At the later stage of the chase, there were CD59 positive puncta and tubular profiles outside Giantin-rings, which were likely Golgi-derived exocytic transport carriers (Figure 5A, arrows in 60 min). Similarly, in live-cell super-resolution imaging, RUSH reporter mCherry-GPI started to appear in the interior of the Golgin84-ring 6 min after chase; it remained there for >30 min before disappearing due to post-Golgi exocytic trafficking (Figure 5C; Figure 5—video 1). Transmembrane RUSH reporters, E-cadherin, VSVG and CD8a-Furin, and a soluble secretory reporter, signal peptide fused GFP, followed similar lateral localization pattern during their intra-Golgi trafficking (Figure 5—figure supplement 1A–D). Collectively, our data demonstrated that conventional secretory cargos partition to the interior of the cisternae during their Golgi transition.

Figure 5 with 4 supplements see all
Conventional or small size secretory cargos can localize to the cisternal interior while bulky ones are restricted to the rim during their intra-Golgi transport.

(A,B) CD59 localizes to the cisternal interior during its intra-Golgi transport. Cells transiently expressing RUSH reporter, SBP-GFP-CD59, were chased in the presence of biotin for indicated time (A). Arrows indicate budding membrane carriers. In (B), the LQ vs time plot measured from the same experiment demonstrated the intra-Golgi transport of CD59. (C) Live-cell imaging showing the interior localization of mCherry-GPI during its transition through the Golgi mini-stack. Cells transiently co-expressing RUSH reporter, SBP-mCherry-GPI, and GFP-Golgin84 were chased in biotin and imaged live under Airyscan super-resolution microscopy. The boxed region in the upper image, which was acquired before the chase, is selected to show the time series. Arrow heads indicate the interior localization. See also Figure 5—video 1. (D–F) The partition of collagenX and mCherry-GPI to the cisternal rim and interior respectively during their intra-Golgi transport. Cells transiently co-expressing RUSH cargos, SBP-GFP-collagenX and SBP-mCherry-GPI were chased as in (A). Arrows and arrow heads indicate the cisternal rim and interior localization respectively. The intra-Golgi transport of collagenX and mCherry-GPI was demonstrated by LQ vs time plots measured from the same experiments in (E) and (F). Error bar, mean ± SEM. n, the number of Golgi mini-stacks used for the calculation. (G) GFP-FM4-CD8a partitions to the cisternal rim upon aggregation. NRK cells transiently expressing GFP-FM4-CD8a were subjected to a combination of D/D solubilizer treatment and wash out at either 20°C or 37°C, as indicated. First set of images is the negative control showing that aggregated GFP-FM4-CD8a was not exported from the ER. Aggregated GFP-FM4-CD8a partitioned to the rim (arrows), while non-aggregated form was still interior-localized (arrow heads). Scale bars represent 500 nm unless specified otherwise.

https://doi.org/10.7554/eLife.41301.015

The secretory cargo wave does not seem to grossly affect the interior distribution of Golgi enzymes, as evidenced by ST6Gal1 (Figure 5—figure supplement 2). By image quantification, >85% of ST6Gal1-moxGFP was found to remain in the interior during the Golgi transition of synchronized VSVG, although a small fluctuation (<4%) was noticed (Figure 5—figure supplement 2A–C). Our finding is different from a previous EM study, in which the shift of Golgi enzymes from the rim to the interior was observed under a traffic wave (Kweon et al., 2004). A more systematic investigation is required to resolve this discrepancy.

Bulky size prevents the localization of secretory cargos at the cisternal interior

Based on EM data, Rothman lab previously proposed that large secretory protein aggregates are segregated to the cisternal rim (Lavieu et al., 2013). To investigate if bulky cargos partition to the rim, we imaged the RUSH reporter GFP-collagenX, a soluble secretory protein that tends to form oligomers (Kwan et al., 1991), by Airyscan super-resolution microscopy. We observed that Golgi-transiting GFP-collagenX appeared either diffuse or punctate (Figure 5D). Assuming that Golgi-localized GFP-collagenX puncta were single multimeric aggregates, using GFP-tagged nucleoporin Nup133 as an in vivo GFP fluorescence standard, we estimated that Golgi-transiting GFP-collagenX puncta had 190 ± 20 copies (n = 77) (Figure 5—figure supplement 3A,B). The diffused collagenX is probably in a much lower oligomeric state. Throughout its intra-Golgi trafficking, collagenX, either in punctate or diffuse appearance, was excluded from the interior of Giantin-rings, where co-expressed mCherry-GPI clearly localized (Figure 5D–F). Instead, it always resided at the rim, either colocalizing with Giantin or surrounding Giantin-rings as discrete puncta. At later stages, the puncta outside Giantin-rings were probably exocytic carriers targeting to the PM.

We also tested soluble and transmembrane secretory cargos, FM4-moxGFP and GFP-FM4-CD8a, whose aggregation states can be controlled by the small molecule — D/D solubilizer. These two cargos are similar to the ones used previously (Lavieu et al., 2013). NRK cells expressing either cargo were treated with D/D solubilizer at 20°C for 2 hr to accumulate and arrest the de-aggregated chimera at the Golgi mini-stack. At 20°C, cells were subsequently subjected to 2 hr of incubation in the presence or absence of D/D solubilizer to either de-aggregate or aggregate the cargo respectively (nocodazole was in the system throughout the procedure). Our previous work has established that secretory cargos such as VSVG are mostly arrested at the medial Golgi under 20°C treatment (Tie et al., 2016b). In some experiments, 10 min warm up at 37°C was conducted before imaging. Using this protocol, the re-aggregated GFP-FM4-CD8a and FM4-moxGFP Golgi puncta upon D/D washout were estimated to have 830 ± 30 (n = 184) and 660 ± 50 (n = 127) copies, respectively (Figure 5—figure supplement 3C,D). We observed that, when in the de-aggregated state, both soluble and membrane FM4-chimeras localized to the interior of Giantin-rings (Figure 5G; Figure 5—figure supplement 3E). Intriguingly, once aggregated, they partitioned to the rim as discrete puncta. Therefore, our light microscopic data indicated that large cargos preferentially partition to the cisternal rim, possibly due to their bulky sizes, while conventional or small cargos tend to locate to the interior.

Discussion

It poses a great challenge to investigate the structure and organization of the Golgi complex by the light microscopy. We established a method to identify the cisternal rim and interior by taking advantage of rim-localized Golgi markers. In addition to quantitative axial localization using the LQ (Tie et al., 2016b), we further showed the advantage of nocodazole-induced Golgi mini-stacks in elucidating the molecular organization of the Golgi complex. We analyzed dozens of Golgi residents representing diverse families of proteins for their lateral localizations. The distribution of enzymes is restricted to the interior of the medial and trans-cisternae. In contrast, trafficking machinery components appear to complement Golgi enzymes by residing at the rim of medial and trans-cisternae, entire cis-Golgi cisternae and trans-Golgi/TGN. Previous EM studies on lateral localizations of trafficking machinery components, including COPI (Orci et al., 1997), giantin (Koreishi et al., 2013), KDEL receptor (Cosson et al., 2002; Martinez-Menárguez et al., 2001; Orci et al., 1997), GS27 (Cosson et al., 2005) and GS15 (Cosson et al., 2005), Golgi enzymes, including Man1B1 (Rizzo et al., 2013), ManII (Cosson et al., 2002; Cosson et al., 2005; Martinez-Menárguez et al., 2001; Orci et al., 2000), MGAT1 (Orci et al., 2000) and GalT (Cosson et al., 2005), and Golgi-transiting cargos including VSVG (Martinez-Menárguez et al., 2001; Mironov et al., 2001) and soluble aggregated FM4-fusion protein (Volchuk et al., 2000), which are summarized and compared in Supplementary file 1 and 2, are mostly consistent with our observations. Our qualitative and quantitative data sketch a Golgi mini-stack as spindle-shaped with medial-cisternae possessing a larger diameter than both cis- and trans-cisternae (Figure 4J). Our morphological description of the Golgi mini-stack, such as the spindle shape of the stack and organization of the TGN, bear similarities to the plant Golgi mini-stack observed by electron tomography (Staehelin and Kang, 2008), probably due to the lack of microtubule cytoskeleton in plants, which is similar to nocodazole-treated mammalian cells. Our findings suggest the spatial partition of the processing and transport function to the interior and rim of the Golgi stack, as depicted by our model in Figure 6.

A schematic model summarizing the organization of a Golgi mini-stack.

LQs of various Golgi residents (see Table 1) are overlaid onto a simplified diagram of a Golgi mini-stack together with the ERES and ERGIC. The red circle represents the mean of the LQ with flanking black bars representing the SEM. The cisternal interior, including central disks and inner-rings, is shaded yellow while the periphery of the Golgi mini-stack, including the cisternal rim, is shaded blue. Within the plot, red circles representing Golgi enzymes (labeled orange at the x-axis) are overlaid onto the yellow-shaded interior region, while those of components of the transport machinery (labeled black at the x-axis) are outside the mini-stack to indicate their periphery localization.

https://doi.org/10.7554/eLife.41301.020

EM studies have revealed that cisternal rims are dilated with a width of ~100 nm, while their stacked interiors are narrow and tightly spaced with a width of ~20 nm (Bykov et al., 2017; Engel et al., 2015; Staehelin and Kang, 2008). Recently, zipper-like intracisternal and intercisternal protein arrays have been discovered at interior regions of medial and trans-cisternae in green alga through the cryo-electron tomography (Engel et al., 2015). It was proposed that these tightly packed protein arrays comprise Golgi enzymes. Our super-resolution and EM data from the Golgi mini-stack provide direct evidence supporting this hypothesis. These enzyme-arrays might organize as an ‘enzyme matrix’ to 1) stack cisternal membrane, 2) retain Golgi enzymes or accessory proteins and 3) exclude trafficking machinery components by a possible molecular crowding mechanism. Therefore, it seems that, collectively, Golgi enzymes determine and maintain the characteristic structure of the Golgi complex.

Most secretory cargos in higher eukaryotes undergo glycosylation in the Golgi complex. Our finding that the cisternal interior and rim correspond to processing and transport domain, respectively, implies that secretory cargos must access interior domains of different cisternae and then reside there long enough for sequential glycosylation. This is indeed what we observed for conventional cargos, such as GPI-anchored proteins, Furin, E-cadherin, VSVG and secretory GFP. On the other hand, these cargos probably have a sufficiently short residence time in the cisternal rim, in which they are either retrieved and retained by the ‘enzyme matrix’ to the interior or packed into membrane carriers targeting to the PM at the trans-Golgi. It seems that the retention by the ‘enzyme matrix’ occurs by default and is independent of glycosylation because secretory GFP is preferentially found within the cisternal interior. However, this is not the case for bulky cargos, such as collagenX and aggregated GFP-FM4-CD8a and FM4-moxGFP, which localized only at the rim and were excluded from the interior. These observations suggest that bulky cargos might be incompatible with the crowded molecular environment of the tightly packed ‘enzyme matrix’ and/or the narrow luminal space at the interior, which can have a width of <10 nm (Engel et al., 2015). Rim partitioning of large secretory cargos has previously been noted by EM (Bonfanti et al., 1998; Engel et al., 2015; Lavieu et al., 2013; Volchuk et al., 2000). Here, we directly visualized by light microscopy the size-dependent lateral partitioning of secretory cargos within the Golgi stack.

This study did not attempt to resolve different intra-Golgi trafficking models and our discoveries can be explained by both cisternal progression and stable compartment models or their modified variants. Nonetheless, our findings provide important insight into the structure and organization of the Golgi complex.

Materials and methods

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Cell line
(Homo sapiens)
HeLa cellATCCATCC: CCL-2; RRID:CVCL_0030
Cell line
(Rattus norvegicus)
Normal
rat kidney (NRK)
fibroblast cell
ATCCATCC: CRL-1570; RRID:CVCL_2144
AntibodyGM130
C-terminus (mouse
monoclonal)
BD BiosciencesBD Biosciences: 610822; RRID:AB_398141(1:500)
AntibodyGolgin 245 (mouse monoclonal)BD BiosciencesBD Biosciences: 611280; RRID:AB_398808(1:100)
AntibodyGGA2 (mouse monoclonal)BD BiosciencesBD Biosciences: 612612; RRID:AB_399892(1:200)
AntibodyGS15 (mouse monoclonal)BD BiosciencesBD Biosciences: 610960;
RRID:AB_398273
(1:250)
AntibodyGS27 (mouse monoclonal)BD BiosciencesBD Biosciences: 611034;
RRID:AB_398347
(1:250)
AntibodyGS28 (mouse monoclonal)BD BiosciencesBD Biosciences: 611184; RRID:AB_398718(1:250)
AntibodySyntaxin6 (mouse monoclonal)BD BiosciencesBD Biosciences:
610635; RRID:AB_397965
(1:250)
AntibodyVti1a (mouse monoclonal)BD BiosciencesBD Biosciences: 611220; RRID:AB_398752(1:500)
AntibodyMyc (mouse monoclonal)Santa cruz biotechnologySanta cruz: sc-40; RID:AB_627268(1:200)
AntibodyCLCB (mouse monoclonal)Santa cruz biotechnologySanta cruz: sc-376414; RRID:AB_11149726(1:200)
AntibodyγCOP (mouse monoclonal)Santa cruz biotechnologySanta cruz:sc-393977; RRID:AB_2753138(1:200)
AntibodyFurin (rabbit polyclonal)Thermo Fisher ScientificThermo Fisher Scientific: PA1062; RRID:AB_2105077(1:100)
AntibodyCI-M6PR (mouse monoclonal)Thermo Fisher ScientificThermo Fisher Scientific: MA1066; RRID:AB_2264554(1:200)
AntibodyAlexa Fluor 594 conjugated streptavidinThermo Fisher ScientificThermo Fisher Scientific: S11227; RRID:AB_2313574(1:500)
AntibodyβCOP (mouse monoclonal)Sigma-AldrichSigma-Aldrich: G6160; RRID:AB_477023(1:200)
AntibodyFlag (mouse monoclonal)Sigma-AldrichSigma-Aldrich: F3165; RRID:AB_259529(1:200)
AntibodyGM130 N-terminus (rabbit monoclonal)AbcamAbcam: ab52649; RRID:AB_880266(1:500)
AntibodyGiantin N-terminus (rabbit polyclonal)BioLegendBiolegend: 924302; RRID:AB_2565451(1:1000)
AntibodyGiantin C-terminus (rabbit polyclonal)this paper(1:500); rabbit polyclonal; against aa3131–3201
AntibodyKDEL receptor (mouse monoclonal)Enzo Life SciencesEnzo Life Sciences: VAA-PT048D; RRID:AB_1083549(1:250)
AntibodyGALNT1Other(1:10); H Clausen lab (University of Copenhagen)
AntibodyGALNT2Other(1:10); H Clausen lab (University of
Copenhagen)
AntibodyArl1 (rabbit polyclonal)PMID: 11792819(1:100)
AntibodyGolgin97 (rabbit polyclonal)PMID: 12972563(1:1000)
Recombinant
DNA reagent
pDMyc-neo-N1this paperSee Supplementary file 3
Recombinant DNA reagentpDMyc-NeoPMID: 12972563
Recombinant
DNA reagent
pGEBPMID: 11792819
Recombinant DNA reagentpA2E-N1PMID: 27369768
Recombinant DNA reagentpmCherry-C2this paperSeeSupplementary file 3
Recombinant DNA reagentStreptavidin-HisPMID: 16554831RRID:Addgene_20860Addgene plasmid #20860
Recombinant DNA reagentStrep-Ii_VSVG-SBP-EGFPPMID: 22406856RRID:Addgene_65300Addgene plasmid #65300
Recombinant DNA reagentss-Strep-KDEL_ManII-SBP-GFPPMID: 22406856RRID:Addgene_65252Addgene plasmid #65252
Recombinant DNA reagentss-Strep-KDEL_ss-SBP-mCherry-GPIPMID: 22406856RRID:Addgene_65295Addgene plasmid #65295
Recombinant DNA reagentTPST1-GFPPMID: 18522538RRID:Addgene_66617Addgene plasmid #66617
Recombinant DNA reagentTPST2-GFPPMID: 18522538RRID:Addgene_66618Addgene plasmid #66618
Recombinant DNA reagentpmScarlet-Giantin-C129PMID: 27869816RRID:Addgene_85048Addgene plasmid #85048
Recombinant DNA reagentli-Strep_ss-SBP-GFPthis paperRUSH reporter of soluble SBP-GFP
Recombinant DNA reagentStrep-Ii_VSVG-SBP-Flagthis paperRUSH reporter of VSVG-SBP-Flag
Recombinant DNA reagentss-Strep-KDEL_ss-SBP-GFP-E-cadherinPMID: 22406856RUSH reporter of SBP-GFP-E-cadherin; a gift from F. Perez lab (Institut Curie)
Recombinant
DNA reagent
ss-Strep-KDEL_ss-SBP-GFP-CD8a-FurinPMID: 26764092RUSH reporter of SBP-GFP-CD8a-Furin
Recombinant
DNA reagent
ss-Strep-KDEL_ss-SBP-GFP-CD59PMID: 26764092RUSH reporter of SBP-GFP-CD59
Recombinant
DNA reagent
ss-Strep-KDEL_ss-SBP-GFP-collagenXOtherRUSH reporter
of SBP-GFP-collagenX; a gift
from F Perez lab
(Institut Curie)
Recombinant
DNA reagent
Rab1a-GFPthis paperSeeSupplementary file 3
Recombinant
DNA reagent
Furin-GFPthis paperSee Supplementary file 3
Recombinant
DNA reagent
Fuin-Mycthis paperSee Supplementary file 3
Recombinant
DNA reagent
GFP-GCC185this paperSeeSupplementary file 3
Recombinant
DNA reagent
GFP-GCC185-mCherrythis paperSeeSupplementary file 3
Recombinant
DNA reagent
GFP-ACBD3this paperSeeSupplementary file 3
Recombinant
DNA reagent
GFP-Rab6this paperSeeSupplementary file 3
Recombinant
DNA reagent
mCherry-Golgin84this paperSeeSupplementary file 3
Recombinant
DNA reagent
GFP-GGA1this paperSeeSupplementary file 3
Recombinant
DNA reagent
mCherry-GM130this paperSeeSupplementary file 3
Recombinant
DNA reagent
Arf1-GFPPMID: 16890159A gift from
FJM van
Kuppeveld
lab (Utrecht
University)
Recombinant
DNA reagent
Arf4-GFPOtherA gift from
FJM van
Kuppeveld
lab (Utrecht
University)
Recombinant
DNA reagent
Arf5-GFPOtherA gift from
FJM van
Kuppeveld lab
(Utrecht University)
Recombinant
DNA reagent
GFP-ERGIC53PMID: 15632110A gift from H Hauri lab (University of Basel)
Recombinant
DNA reagent
GFP-GM130PMID: 11781572A gift from M De Matties lab (Telethon Institute of Genetics and Medicine, Italy)
Recombinant
DNA reagent
GFP-Golgin84PMID: 12538640A gift from M Lowe lab (University of Manchester)
Recombinant
DNA reagent
GFP-Golgin97PMID: 11792819A gift from W Hong lab (Institute of Molecular and Cell Biolgoy, Singapore)
Recombinant DNA reagentGPP130-GFPPMID: 9201717A gift from
A Linstedt lab (Carnegie Mellon University)
Recombinant
DNA reagent
GRASP55-GFPOtherA gift from Y Zhuang lab (University of Michigan)
Recombinant DNA reagentGRASP65-GFPOtherA gift from Y Zhuang lab (University of Michigan)
Recombinant
DNA reagent
DMyc-GCC185OtherA gift from W Hong lab (Institute of Molecular and Cell Biolgoy, Singapore)
Recombinant
DNA reagent
Sec23a-mCherryOtherA gift from W Hong lab (Institute of Molecular and Cell Biolgoy, Singapore)
Recombinant DNA reagentSec31a-GFPPMID: 10788476A gift from W Hong lab (Institute of Molecular and Cell Biolgoy, Singapore)
Recombinant
DNA reagent
Vamp4-GFPPMID: 17327277A gift from W Hong lab (Institute of Molecular and Cell Biolgoy, Singapore)
Recombinant DNA reagentMyc-Sec34PMID: 11929878A gift from
W Hong lab
(Institute of
Molecular and
Cell Biolgoy,
Singapore)
Recombinant
DNA reagent
Myc-Sec13PMID: 22609279A gift from W
Hong lab
(Institute of Molecular
and Cell Biolgoy,
Singapore)
Recombinant DNA reagentMGAT1-AcGFP1this paperSee Supplementary file 3
Recombinant DNA reagentMGAT2-AcGFP1this paperSee Supplementary file 3
Recombinant DNA reagentMGAT4B-AcGFP1this paperSee Supplementary file 3
Recombinant DNA reagentST6Gal1-AcGFP1this paperSee Supplementary file 3
Recombinant DNA reagentMan1B1-Mycthis paperSee Supplementary file 3
Recombinant DNA reagentMGAT1-Mycthis paperSee Supplementary file 3
Recombinant DNA reagentMGAT2-Mycthis paperSee Supplementary file 3
Recombinant DNA reagentST6Gal1-Mycthis paperSee Supplementary file 3
Recombinant DNA reagentβ4GalT3-Mycthis paperSee Supplementary file 3
Recombinant DNA reagentGalT-mCherryPMID: 26764092
Recombinant DNA reagentSLC35C1-MycOriGene Technologies Inc.Cat. No.: RC200101
Recombinant DNA reagentβ3GalT6-MycOriGene Technologies Inc.Cat. No.: MR204731
Recombinant DNA reagentβ4GalT7-MycOriGene Technologies Inc.Cat. No.: RC200258
Recombinant DNA reagentPOMGNT1-MycOriGene Technologies Inc.Cat. No.: RC200176
Recombinant DNA reagentFM4-moxGFPthis paperSee Supplementary file 3
Recombinant DNA reagentGFP-FM4-CD8aPMID: 23755362A gift from James Rothman lab (Yale University)
Recombinant DNA reagentGPP130-APEX2-GFPthis paperSee Supplementary file 3
Recombinant DNA reagentMGAT2-APEX2-GFPthis paperSee Supplementary file 3
Recombinant DNA reagentHis-Giantin(3131–3201)this paperSee Supplementary file 3
Recombinant DNA reagentGST-Giantin(3131–3235)this paperSee Supplementary file 3
Recombinant DNA reagentGFP-Nup133-mutPMID: 27613095See Supplementary file 3
Recombinant DNA reagentshNup133-1PMID: 27613095See Supplementary file 3
Commercial assay or kitAPEX Alexa Fluor 488 Antibody Labeling KitThermo Fisher ScientificInvitrogen: A10475
Commercial assay or kitAPEX Alexa Fluor 488 Antibody Labeling KitThermo Fisher ScientificInvitrogen A10468
Chemical compound, drugbiotinIBAIBA: 2101600240 μM
Chemical
compound, drug
biotin phenolIris Biotech GmbHIris Biotech GmbH: LS3500500 μM
Chemical compound, drugnocodazoleMerckMerck: 48792833 μM
Chemical compound, drugD/D solubilizerClontechClontech: 6350541 mM
Software,
algorithm
FijiPMID: 22743772https://fiji.sc/
Software,
algorithm
Calculation of the LQPMID: 26764092; PMID: 28829416
Software,
algorithm
Gyradius and intensity normalization.ijmthis paperTo normalize diameters and intensities of en face Golgi mini-stacks
Software,
algorithm
Golgi mini-stack alignment.ijmthis paperTo align normalized en face Golgi mini-stacks
Software,
algorithm
Radial mean intensity profile.ijmthis paperTo measure radial mean intensity of en
face averaged Golgi mini-stacks

DNA plasmids

See Supplementary file 3.

Antibodies and small molecules

The following mouse monoclonal antibodies (mAbs) were purchased from BD Biosciences: GM130 C-terminus, Golgin245, GGA2, GS15, GS27, GS28, Syntaxin6 and Vti1a. The following mouse mAbs were from Santa Cruz: Myc, CLCB and γCOP. Rabbit polyclonal antibody (pAb) against Furin, mouse mAb against CI-M6PR and Alexa Fluor 594 conjugated streptavidin were from Thermo Fisher Scientific. The following antibodies were commercially available from respective vendors: mouse mAb against Flag-tag and βCOP (Sigma-Aldrich), rabbit mAb against the N-terminus of GM130 (Abcam), rabbit pAb against Giantin (BioLegend) and mouse mAb against KDEL receptor (Enzo Life Sciences). Mouse mAbs against GALNT1 and GALNT2 were from H. Clausen. Rabbit pAbs against Arl1 and Golgin97 were previously described (Lu and Hong, 2003; Lu et al., 2001). The following small molecules were commercially available: biotin (IBA), biotin phenol (Iris Biotech GmbH), nocodazole (Merck) and D/D solubilizer (Clontech).

Cell lines

HeLa and normal rat kidney fibroblast (NRK) cells were from American Type Culture Collection. Cell were assumed to be authenticated by the supplier. The presence of mycoplasma contamination was monitored by Hoechst 33342 staining.

Cell culture and transfection

HeLa and NRK cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum. Cell transfection was conducted using Orci et al., 2000 (Invitrogen) according to manufacturer’s manual. In live-cell imaging, cells grown on a Φ35 mm glass-bottom Petri-dish (MatTek) were imaged in CO2-independent medium (Invitrogen) supplemented with 4 mM glutamine and 10% fetal bovine serum at 37°C. Unless otherwise indicated, all cells used were HeLa and treated with 33 µM nocodazole to induce the formation of Golgi mini-stacks.

Production of Giantin C-terminal antibody

It was conducted as previously described (Madugula and Lu, 2016; Mahajan et al., 2013). Briefly, His-Giantin(3131–3201) was purified in urea from bacteria and used as the antigen to raise the anti-serum in rabbits (Genemed Synthesis Inc). Recombinant GST-Giantin(3131–3235) was purified from bacteria and subsequently used to purify the antibody from the anti-serum.

Super-resolution fluorescence microscopy

The Airyscan super-resolution microscope system (Carl Zeiss) comprises a Zeiss LSM710 confocal microscope equipped with an oil objective lens (alpha Plan-Apochromat 100 ×, 1.46 NA), a motorized stage, a temperature control environment chamber and Airyscan module. Fluorophores were excited by three laser lines with wavelengths of 488, 561 and 640 nm and their respective emission bandwidths were 495–550 nm, 595–620 nm and long pass 645 nm. The microscope system was controlled by ZEN software (Carl Zeiss). Pixel size of images ranged from 40 to 54 nm. For 3D imaging, the z-step of image stacks was 170 nm. Image stacks were subjected to Airyscan processing and maximal intensity projection (MIP) in ZEN software. Image analysis was performed in Fiji (https://imagej.net/Fiji). We exhausted our images for all Golgi mini-stacks that were visually identifiable as either en face or side views.

En face averaging of golgi mini-stack images and radial mean intensity profile acquisition

En face view images of Giantin-labeled Golgi mini-stacks were averaged in semi-automatic software tools that were developed using macros of Fiji. Mini-stack images were first cropped to square shape and subjected to background subtraction. To quantify the size of the Giantin-ring, we adopted the concept of the gyradius from physics. For pixel i in the Giantin-ring image, assuming that Ii is its intensity and ri is its distance to the center of fluorescence mass, the gyradius of the Giantin-ring can be calculated as

(Iiri2)Ii,

with all pixels of the image considered. The macro ‘gyradius and intensity normalization’ (see Source code 1) calculates the gyradius of Giantin in a set of multi-channel images and resizes the set of images so that the gyradius of Giantin is 100 pixels. The canvas of the image set is further expanded to 701 × 701 pixel. Using the macro ‘Golgi mini-stack alignment’ (see Source code 2), Golgi marker images are aligned so that their centers of fluorescence mass are at (350, 350), the center of the image. The en face averaged Golgi mini-stack image is acquired by z-projection of these aligned images. The radial mean intensity profile is acquired using the macro ‘Radial mean intensity profile’ (see Source code 3). The mean intensity of all pixels within a circle around the center of the fluorescence mass is plotted against its radius (ranging from 1 to 350 pixels). The radius of a Golgi marker is defined by the half maximum position of its outer slope of the intensity plot and is normalized by the corresponding radius of Giantin. Detailed steps are described in Supplementary file 4.

Measuring diameters of Giantin-rings

To measure the diameter of a Giantin-ring, a line was first drawn across its center. In the resulting line intensity profile (Fiji), the diameter of the ring was defined as the distance between the two half-maximum-intensity points at outer slopes.

Immunofluorescence labeling and RUSH cargo trafficking assay

These were conducted as previously described (Tie et al., 2016b). By default, tagged-proteins were transiently transfected while non-tagged proteins were native and immuno-stained by their antibodies.

Fluorescence labeling of APEX2-mediated biotinylation

Nocodazole-treated HeLa cells expressing MGAT2-APEX2-GFP were incubated with 500 μM biotin phenol for 30 min at 37°C. Cells were subsequently transferred to ice and treated with 1 mM H2O2 for 1 min with brief agitation. After extensive washing with PBS containing 10 mM sodium ascorbate (Sigma-Aldrich), 5 mM Trolox (Sigma-Aldrich), and 10 mM sodium azide (Sigma-Aldrich), cells were fixed and processed for immunofluorescence. Biotinylated proteins were labeled by Alexa Fluor 594 conjugated streptavidin.

APEX2-EM

EM was performed as previously described (Ludwig et al., 2016) with minor modifications. In brief, NRK cells transiently expressing GPP130-APEX2-GFP or MGAT2-APEX2-GFP were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 (CB) containing 2 mM CaCl2 for 1 hr on ice, rinsed three times in CB, and incubated in 0.5 mg/ml 3,3’-diaminobenzidine and 0.5 mM H2O2 in CB for 5 min. Cells were washed several times in CB and post-fixed in 1% osmium tetroxide in CB containing 2 mM CaCl2 supplemented with 1% (w/v) potassium ferricyanide for 1 hr on ice in the dark. Samples were further processed as described previously (Ludwig et al., 2016). After image acquisition, only Golgi stacks with long axis >500 nm were analyzed.

Calculation of the LQ

The LQ of a Golgi protein was acquired as previously described using a conventional wide-field fluorescence microscope (Tie et al., 2017; Tie et al., 2016b).

Estimating the stoichiometry of the fluorescence protein aggregate

This was performed using our previously established method (Tie et al., 2016a). HeLa cells were co-transfected with shNup133-1 and GFP-Nup133-mut to knock down the endogenous Nup133 and replace it with shRNA-resistant GFP-Nup133-mut. The resulting nuclear pores, which contain ~16 GFP-Nup133-mut (Tie et al., 2016a), were used as a fluorescence standard to quantify the copy number of GFP-collagenX, GFP-FM4-CD8a and FM4-moxGFP at Golgi-localized puncta. Identical imaging conditions were used under Airyscan super-resolution microscopy to image Nup133 and the fluorescence protein aggregate puncta. In GFP-Nup133-mut image, a circular region of interest (ROI) that contains a nuclear pore was generated and its total intensity was quantified as INup (Tie et al., 2016a). The total intensity of a circular ROI containing a Golgi punctum was also similarly acquired as Ipunctum. The copy number of GFP-tagged chimera in the Golgi punctum was therefore calculated as 16 × Ipunctum/ INup. To quantify the copy number of FM4-moxGFP, moxGFP was assumed to be 1.47-fold brighter than EGFP (https://www.addgene.org/fluorescent-proteins/plasmid-backbones/), which is called GFP in this study, and the copy number of FM4-moxGFP in the Golgi punctum was calculated as 10.9 × Ipunctum/ INup.

Fluorescence-conjugation of Giantin antibodies

Alexa Fluor 647 and Alexa Fluor 488 were covalently conjugated onto a commercial (BioLegend) (against the N-terminus) and our homemade (against the C-terminus) rabbit pAb against Giantin, respectively, using APEX antibody labeling kit (Invitrogen) according to the manufacturer’s protocol.

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Decision letter

  1. Suzanne R Pfeffer
    Reviewing Editor; Stanford University, United States
  2. Anna Akhmanova
    Senior Editor; Utrecht University, Netherlands

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "The spatial separation of processing and transport functions to the interior and periphery of the Golgi stack" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Suzanne R Pfeffer as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Anna Akhmanova as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Frederic A Bard (Reviewer #2); Alberto Luini (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

This is a high quality description of the spatial distribution of Golgi enzymes and trafficking machinery in nocodazole-induced Golgi ministacks using super resolution light microscopy and the RUSH system to monitor transit through the compartment. The authors find that glycosyltransferases appear to reside in the internal portion of the cisternae while trafficking machinery can be found at the rims. The work will be a valuable resource to readers of eLife if it was made more scholarly and quantification is added. We suggest that the revision be submitted in the category of a Tools and Resources article.

Most of the suggested edits are textual but important. Two experiments and some additional quantifications are required for revision.

1) The authors use different conditions to study the distribution of enzymes and of cargo proteins. They examine the former in unperturbed cells, and the latter during a traffic wave. However, intense traffic could alter the distribution of the enzymes. Please examine the distribution of at least a couple of enzymes during a traffic pulse. The results might be very interesting and will add something novel to the story.

2) The EM is of low quality and needs quantification. All EM localizations need to be quantified with a suitable N value by some metric to convince the reader of enzyme distributions across the stacks. In addition, the authors conclude that "the total amount of GnT2 in these vesicles was probably much less than that in the interior since we did not find APEX2-signal outside Giantin-rings by fluorescence imaging of Golgi mini-stacks". APEX is non-linear in terms of the signal it generates and so one must be careful about concluding too much from an amplified signal. The fact that the authors did not find the APEX2-signal of GnT2 outside giantin-rings by fluorescence does not mean that GnT2 is less in vesicles than the interior. Only immunogold can tell you absolute amounts of protein, but we are not asking you to carry out immuno EM for this study.

Other comments:

1) In Figure 4A and 4D, the authors show the peripheral and central distribution of small and bulky cargos during the intra Golgi-transport. The time points they study are not appropriate.

It has been extensively shown that a secretory cargo crosses the Golgi within 12-15 minutes. Their synchronization protocol does not allow one to understand what stage of intra-Golgi traffic one is observing. It might be more productive to use the 15 degree block protocol and follow cargo transport along the cis to trans axis in the next 15 min.

2) The authors conclude that "large cargos preferentially partition to the cisternal rim, possibly due to their bulky sizes, while conventional or small cargos tend to locate to the interior". A problem here is that transport is synchronized at 20 °C, which is well known to arrest the secretory proteins mainly in the TGN. The TGN is morphologically and functionally different from the Golgi cisternae, and when cargo reaches the TGN, the intra Golgi transport is complete. This experiment is confusing and needs to be described with greater care.

3) Many of the proteins they study are tagged and overexpressed. This is probably necessary for many enzymes, whose endogenous levels are very low. However, overexpression might alter their distribution. This cannot really be fixed but it should be stated clearly and discussed – Orci demonstrated this in previous EM studies.

4) For the Golgi images for the various markers, the line scans reflect individual cells. It would be important to include quantitation of the number of cells showing a particular staining pattern, in addition to selected images. Figure 3G shows a summary but we could not find any values of N for the experiments. Perhaps for example markers, the localizations of 30 cells could be overlaid or a metric used to distinguish donut morphology from spheres? More quantitation is important.

5) The authors need to be more scholarly about previous immuno EM localizations of various Golgi enzymes and trafficking machinery in relation to their own story. Quantitative work of Orci and Klumperman for example, need to be referred to in a table in comparison with the present data. There is also lots of published information regarding glycosyltransferase localization at the EM level. Subsection “Glycosylation enzymes reside at the interior of a Golgi stack”, first paragraph, this was in plants, which build entirely different glycans. Please use mammalian cell references for the precise enzymes studied – and note in the text where this method confirms.

6) Subsection “trans-Golgi and TGN proteins (LQ ≥ 1.0)”. In most cell types, CI-M6PR (and possibly Furin) is in perinuclear late endosomes next to the Golgi, which would explain lack of co-localization with TGN markers. The clathrin stain has been shown previously to be in a distinct TGN microdomain (Brown et al., 2011). Please correct the text.

7) In the Discussion section, they suggest that the enzymes might be a major component of the protein intracisternal matrix that has been previously visualized by other authors by EM. But this is very unlikely to be the case, as Golgi enzymes are expressed in very low copy numbers.

They venture into speculation about the transport mechanism within the Golgi. It is obvious that the distributions at steady state of cargo proteins and enzymes does not allow them to distinguish between transport models, and the authors actually state that they do not want to speculate on their observations in this regard. In the previous paragraph, however, they suggest that the observed lateral distributions can be linked to anterograde vesicular movement of the cargo proteins which, once deposited in the cisterna would reach the central domain of the cisterna to be glycosylated by enzymes residing in that region. This is an interpretation in favor of the anterograde vesicular transport model. Here the authors draw conclusions that are not based on data and at the same time contradict themselves. Please adhere to a simpler unbiased logic in the Discussion.

8) It seems that the sentence "conventional secretory cargos were observed to transit the cisternal interior before exiting the Golgi at the rim" in the Abstract is more an extrapolation from the data than an observed phenomenon. Since it bears on the debate of cisternal maturation versus vesicular transport of cargo, please modify the text.

9) It would improve greatly the accessibility of the paper to have a summary model in the form of a 3D graphic. It would also be interesting (although maybe not for this paper) to have a simple website document summarizing the sub-cellular localisations of all these Golgi proteins.

10) It should be discussed that most enzymes studied are related to the N-glycosylation pathway. It should also be explained which enzymes are not part of this pathway. Indeed, most glycosylation enzymes are involved in specific glycosylation pathways and different pathways may have slightly different enzymatic distributions within the Golgi.

11) Is there evidence that bulky cargoes have less elaborated N-glycans than small cargoes?

12) COPII components localisation: is this specific for nocodazole induced mini-stacks? It is known indeed that mini-stacks form near ERES. Do the authors think these values would hold for a normal Golgi?

13) The authors propose that some enzymes have a different cisternal distribution (inner ring for some). Is it possible to compare with data obtained using FRET, notably by the group of Sakari Kellokumpu?

https://doi.org/10.7554/eLife.41301.030

Author response

1) The authors use different conditions to study the distribution of enzymes and of cargo proteins. They examine the former in unperturbed cells, and the latter during a traffic wave. However, intense traffic could alter the distribution of the enzymes. Please examine the distribution of at least a couple of enzymes during a traffic pulse. The results might be very interesting and will add something novel to the story.

This is a very interesting suggestion. We studied the effect of the synchronized trafficking wave of RUSH reporter, VSVG-SBP-Flag, on the lateral distribution of ST6Gal1-moxGFP. HeLa cells co-expressing VSVG-SBP-Flag, ST6Gal1-moxGFP and GalT-mCherry were treated with biotin to chase VSVG and en face averaged images were acquired (the average of size and intensity normalized and center aligned en face images; please see the revised Materials and methods). Cisternal rim ROI was defined by the donut formed by the radius of en face averaged ST6Gal1-moxGFP (not subjected to traffic wave) and that of Giantin-ring. The percentage of ST6Gal1 within the interior was quantified at different chase time (min) and is shown in revised Figure 5—figure supplement 2 with the corresponding plot of VSVG-SBP-Flag’s LQ vs. time from the same experiment.

We did not observe any obvious change of the interior localization of Golgi enzymes. However, it is an important control to demonstrate the interior localization of enzymes under traffic wave. We have inserted the below text to the Results section: “The secretory cargo wave does not seem to grossly affect the interior distribution of Golgi enzymes, as evidenced by ST6Gal1 (Figure 5—figure supplement 2). […] A more systematic investigation is required to resolve this discrepancy.”

2) The EM is of low quality and needs quantification. All EM localizations need to be quantified with a suitable N value by some metric to convince the reader of enzyme distributions across the stacks. In addition, the authors conclude that "the total amount of GnT2 in these vesicles was probably much less than that in the interior since we did not find APEX2-signal outside Giantin-rings by fluorescence imaging of Golgi mini-stacks". APEX is non-linear in terms of the signal it generates and so one must be careful about concluding too much from an amplified signal. The fact that the authors did not find the APEX2-signal of GnT2 outside giantin-rings by fluorescence does not mean that GnT2 is less in vesicles than the interior. Only immunogold can tell you absolute amounts of protein, but we are not asking you to carry out immuno EM for this study.

We have imaged more Golgi stacks and performed a simple statistical analysis by quantifying the percentage of MGAT2-APEX2 and GPP130-APEX2 positive Golgi stacks that display predominant interior and rim distribution, respectively. From almost 60 Golgi stacks imaged for each construct, we found that, for MGAT2, 93% (n=58 from 14 cells) stacks demonstrated interior localization while, for GPP130, 68% (n=57 from 25 cells) stacks displayed rim localization. We have modified the manuscript to include these data.

We agree that the APEX2-catalyzed reaction is non-linear and deleted the conclusion that “the total amount of GnT2 in these vesicles was probably much less than that in the interior”. Below is the revised corresponding text:

“Noticeably, APEX2-generated electron density was also found in vesicles and budding profiles at the rim (arrow heads in Figure 4I). However, we did not find MGAT2 (Figure 1K and 4C) or MGAT2-APEX2 (Figure 4—figure supplement 1U) signal outside Giantin-rings by fluorescence imaging of Golgi mini-stacks.”

Other comments:

1) In Figure 4A and 4D, the authors show the peripheral and central distribution of small and bulky cargos during the intra Golgi-transport. The time points they study are not appropriate.

It has been extensively shown that a secretory cargo crosses the Golgi within 12-15 minutes. Their synchronization protocol does not allow one to understand what stage of intra-Golgi traffic one is observing. It might be more productive to use the 15 degree block protocol and follow cargo transport along the cis to trans axis in the next 15 min.

In Figure 4A and 4D of previous version manuscript, our protocol synchronizes the traffic of secretory cargos from the ER. This should not be an issue since we also showed (in Figure 4B, 4E and 4F) plots of LQ vs. time measured from the same experimental data sets. LQ vs. time plots very accurately and numerically indicate the position of secretory cargos within the Golgi stack or the secretory pathway at the time of interest. Take note that LQ is linear and 0.0 and 1.0 correspond to GM130 and GalT-mCherry localizations respectively. Therefore, by combining both en face view imaging and LQ vs. time data (A and B; D and E, F), we are able to tell the lateral distribution (rim or interior) of a cargo when it is at a precise intra-Golgi position along the cis-trans axis of the Golgi mini-stack.

2) The authors conclude that "large cargos preferentially partition to the cisternal rim, possibly due to their bulky sizes, while conventional or small cargos tend to locate to the interior". A problem here is that transport is synchronized at 20 °C, which is well known to arrest the secretory proteins mainly in the TGN. The TGN is morphologically and functionally different from the Golgi cisternae, and when cargo reaches the TGN, the intra Golgi transport is complete. This experiment is confusing and needs to be described with greater care.

In our previous study, we demonstrated that, at 15 and 20°C, the LQ of secretory cargo VSVG-GFP was arrested at −0.06 ± 0.03 (n = 97) and 0.56 ± 0.03 (n= 124), corresponding to the cis- and medial-Golgi localization, respectively (Tie et al., 2016). Our data indicated that at least a significant pool of a small secretory cargo does not reach the trans-Golgi or TGN at 20 °C. The LQ value of 0.56 for VSVG-GFP is likely resulted from the almost even distribution from the cis to trans-Golgi cisternae at 20 °C. Therefore, there should be still intra-Golgi transport after warming up the system to 37 °C.

We added the following sentence to give this background information: “Our previous work has established that secretory cargos such as VSVG are mostly arrested at the medial Golgi under 20 °C treatment (Tie et al., 2016)”.

3) Many of the proteins they study are tagged and overexpressed. This is probably necessary for many enzymes, whose endogenous levels are very low. However, overexpression might alter their distribution. This cannot really be fixed but it should be stated clearly and discussed – Orci demonstrated this in previous EM studies.

The problem of the overexpression on a protein’s subcellular localization is a general and valid concern for all studies. It certainly applies to our study. We think that this reviewer refers to the following paper: Dynamic transport of SNARE proteins in the Golgi apparatus by Cosson et al. (2005), where the authors studied the effect of overexpression of SNAREs on its localization in comparison to endogenous ones. We have modified our text in Results by adding the below sentences: “Due to the lack of reagents to detect endogenous proteins, many residents were detected by the overexpression of their tagged fusions (Table 1). Caution must be taken in the interpretation of our data as it has been documented that overexpression can change both the axial and lateral localization of Golgi residents (Cosson et al., 2005).”.

4) For the Golgi images for the various markers, the line scans reflect individual cells. It would be important to include quantitation of the number of cells showing a particular staining pattern, in addition to selected images. Figure 3G shows a summary but we could not find any values of N for the experiments. Perhaps for example markers, the localizations of 30 cells could be overlaid or a metric used to distinguish donut morphology from spheres? More quantitation is important.

We have developed a software tool to average en face view images of Golgi mini-stacks. Golgi mini-stacks were first co-stained for Giantin and a testing Golgi marker. En face view images of a Golgi marker were subsequently size-normalized by radii of their corresponding Giantin-rings, intensity-normalized to 200 million and aligned according to their centers of fluorescence mass. Only Golgi residents localizing to the stack (LQ in between 0 and 1) were averaged. The en face averaging provides a powerful tool to survey a large number of mini-stack images and extract information such as radius and radial mean intensity profile. The radius of a Golgi marker were normalized by that of Giantin (normalized radius) and plotted against the Golgi marker’s LQ. In revamped Figure 4J (previously Figure 3G), the normalized lateral distribution vs. LQ plot was replaced by normalized radius vs. LQ. The number of mini-stack images used for averaging is indicated by n in Figure 4J. Through en face averaging, we further found that TPST2-GFP displays inner ring appearance. Figures have been modified to include these averaged images and corresponding radial mean intensity profiles.

5) The authors need to be more scholarly about previous immuno EM localizations of various Golgi enzymes and trafficking machinery in relation to their own story. Quantitative work of Orci and Klumperman for example, need to be referred to in a table in comparison with the present data.

EM studies on the Golgi mostly concerned the cis-trans or axial localizations. As suggested by this reviewer, we have examined past quantitative EM studies, especially those from Orci, Luini and Klumperman labs, on the lateral distributions of Golgi residents or Golgi transiting secretory cargos. It is difficult to compare EM results with our light microscopy data since definitions of interior and rim are likely to differ. Our attempt of such comparison is now summarized in Appendix 1.

There is also lots of published information regarding glycosyltransferase localization at the EM level. Subsection “Glycosylation enzymes reside at the interior of a Golgi stack”, first paragraph, this was in plants, which build entirely different glycans. Please use mammalian cell references for the precise enzymes studied – and note in the text where this method confirms.

As suggested, we have added the following sentences to give localization examples of mammalian Golgi enzymes involved in the modification of N-glycan. “This observation is consistent with previous EM studies. […] Second, it has been documented that the sub-Golgi localization of enzymes can be cell-type dependent (Velasco et al., 1993).”

6) Subsection “trans-Golgi and TGN proteins (LQ ≥ 1.0)”. In most cell types, CI-M6PR (and possibly Furin) is in perinuclear late endosomes next to the Golgi, which would explain lack of co-localization with TGN markers. The clathrin stain has been shown previously to be in a distinct TGN microdomain (Brown et al., 2011). Please correct the text.

In native cells (without nocodazole treatment), a significant pool of CI-M6PR localizes to the perinuclear late endosome. Therefore, there are CI-M6PR positive punctate structures near the TGN but they neither co-localize with the TGN nor do they represent carriers or budding profiles directly derived from the TGN, as commented by this reviewer. However, in cells treated with nocodazole, CI-M6PR positive late endosomes are no longer perinuclear and, instead, they localize throughout the cytosol. Therefore, except by chances, late endosomes are not commonly found near Golgi mini-stacks and a majority CI-M6PR punctate structures should be membrane profiles derived from the TGN. Hence, we think that the distinct localization pattern of CI-M6PR can be explained by their localization at the different domain of the TGN from other markers. We have cited the suggested JCB paper by Brown et al. for the microdomain of TGN and Ladinsky et al. (1999) for clathrin microdomains, which are projected away from the Golgi stack.

7) In the Discussion section, they suggest that the enzymes might be a major component of the protein intracisternal matrix that has been previously visualized by other authors by EM. But this is very unlikely to be the case, as Golgi enzymes are expressed in very low copy numbers.

They venture into speculation about the transport mechanism within the Golgi. It is obvious that the distributions at steady state of cargo proteins and enzymes does not allow them to distinguish between transport models, and the authors actually state that they do not want to speculate on their observations in this regard. In the previous paragraph, however, they suggest that the observed lateral distributions can be linked to anterograde vesicular movement of the cargo proteins which, once deposited in the cisterna would reach the central domain of the cisterna to be glycosylated by enzymes residing in that region. This is an interpretation in favor of the anterograde vesicular transport model. Here the authors draw conclusions that are not based on data and at the same time contradict themselves. Please adhere to a simpler unbiased logic in the Discussion.

We are open to all possible models of intra-Golgi trafficking. Thanks for pointing out this discrepancy. We have modified the corresponding text: “On the other hand, these cargos probably have a sufficiently short residence time in the cisternal rim, in which they are either retrieved and retained by the “enzyme matrix” to the interior or packed into membrane carriers targeting to the PM at the trans-Golgi”.

8) It seems that the sentence "conventional secretory cargos were observed to transit the cisternal interior before exiting the Golgi at the rim" in the Abstract is more an extrapolation from the data than an observed phenomenon. Since it bears on the debate of cisternal maturation versus vesicular transport of cargo, please modify the text.

We have modified it to: “conventional secretory cargos appeared at the cisternal interior during their intra-Golgi trafficking and transiently localized to the cisternal rim before exiting the Golgi.”

9) It would improve greatly the accessibility of the paper to have a summary model in the form of a 3D graphic. It would also be interesting (although maybe not for this paper) to have a simple website document summarizing the sub-cellular localisations of all these Golgi proteins.

We have provided a 2D schematic model to summarize the key findings in Figure 6. We will map more sub-Golgi localizations or LQs of Golgi residents and publish these data in certain form in the near future.

10) It should be discussed that most enzymes studied are related to the N-glycosylation pathway. It should also be explained which enzymes are not part of this pathway. Indeed, most glycosylation enzymes are involved in specific glycosylation pathways and different pathways may have slightly different enzymatic distributions within the Golgi.

As suggested, the following text was added to Results to categorize enzymes in our study: “We studied components of Golgi post-translational modification machinery (Table 1; Supplementary file 1), including a GDP-fucose transporter, SLC35C1 (Lubke et al., 2001), and more than a dozen enzymes involved in N-glycosylation (ManIB1, MGAT1, ManII, MGAT2, GalT, SialT and MGAT4B), O-glycosylation (GalNT1, GalNT2 and POMGNT1), poly-N-acetyllactosamine synthesis (β4GalT3), glycosaminoglycan synthesis (β3GalT6 and β4GalT7) and sulfation (TPST1 and 2)”. Therefore, in addition to N-glycosylation enzymes, enzymes involved in other glycosylation and post-translational modifications were also studied.

11) Is there evidence that bulky cargoes have less elaborated N-glycans than small cargoes?

This is an interesting question that we also wanted to address. We reported in this manuscript that bulky cargoes transit through the rim of Golgi cisternae while Golgi enzymes localize to the interior. According to our knowledge, it is currently unclear whether bulky cargoes have less elaborated N- or O-glycosylation than small cargos. We think that the rim partition probably doesn’t abolish the Golgi-type N-glycosylation modification of large secretory cargos. This is because: (1) ManI, MGAT4B and ManII can localize to the rim; (2) minor amount of enzymes could transiently pass through the rim; and (3) the low amount of glycosylation enzymes could be sufficient to modify the low copy numbers of the large secretory cargos in the rim.

As the best known large secretory cargo, collagen has been noted to be extensively O-glycosylated by galactosyl and glucosyl transferases. Interestingly, different from the O-glycosylation at the Golgi, the O-glycosylation of collagen takes place in the ER before its triple helical structure is assembled (Kadler, 1994). Collagen galactosyltransferase contains RDEL ER localization-motif (Schegg et al., 2009) and has been shown to localize to the ER (Liefhebber et al., 2010). However, some collagens, such as Col4a1 and Col4a2, have single N-glycosylation (Basak et al., 2016), suggesting that collagen might complete the N-glycan modification in the Golgi. Another large secretory cargo, high molecular weight adiponectin, similarly has N-glycan in addition to extensive galactosyl and glucosyl O-glycosylation modifications at the ER.

It is probably difficult to design experiments to address this question since the glycosylation process is usually unique for each protein. Hence, it would be a good strategy if the glycosylation of the same protein can be compared between its monomer and aggregated form. One experiment that we can think of is the small molecule inducible aggregation cargos, such as FM4, engineered with artificial glycosylation motifs. However, for a tight aggregate, it is likely that only surface-exposed glycosylation sites can be accessed and modified by enzymes, resulting in a much lower glycosylation efficiency than monomers and thus obscuring result interpretation.

Therefore, although this is a very interesting question that would be a good follow up study, no conclusion can be made at this stage. We therefore did not discuss it in the revised manuscript.

12) COPII components localisation: is this specific for nocodazole induced mini-stacks? It is known indeed that mini-stacks form near ERES. Do the authors think these values would hold for a normal Golgi?

The LQs of COPII components only apply to the nocodazole-induced Golgi mini-stacks. In native cells that are not treated with nocodazole, COPII and ERES are not physically coupled to the Golgi stacks and their distances can vary greatly. However, we think that LQ values still provide important information for the logical organization of the ERES, ERGIC and Golgi.

13) The authors propose that some enzymes have a different cisternal distribution (inner ring for some). Is it possible to compare with data obtained using FRET, notably by the group of Sakari Kellokumpu?

We found that Man1B1, ManII and MGAT4B appear as inner-rings concentric to corresponding Giantin-rings while other N-glycosylation enzymes localize to the central disk. FRET studies from Kellokumpu lab reported that, among Golgi N-glycosylation enzymes they tested, including MGAT1, MGAT2, GalT and ST6Gal1, only MGAT1/2 and GalT/ ST6Gal1 were found to assemble as heteromeric complexes (Hassinen et al., 2010). Our lateral localization data are consistent with the report from Kellokumpu lab as both MGAT1/2 and GalT/ ST6Gal1 complexes localize to the central disk and the heteromeric interaction might contribute to such interior localization.

References:

Basak T, Vega-Montoto L, Zimmerman LJ, Tabb DL, Hudson BG, Vanacore RM. (2016) Comprehensive Characterization of Glycosylation and Hydroxylation of Basement Membrane Collagen IV by High-Resolution Mass Spectrometry. J Proteome Res. 15(1):245-58.

Hassinen A, Rivinoja A, Kauppila A, Kellokumpu S. (2010) Golgi N-glycosyltransferases form both homo- and heterodimeric enzyme complexes in live cells. J. Biol. Chem. 285(23):17771–17777. doi: 10.1074/jbc.M110.103184

Liefhebber JM, Punt S, Spaan WJ, van Leeuwen HC. (2010) The human collagen beta(1- O)galactosyltransferase, GLT25D1, is a soluble endoplasmic reticulum localized protein. BMC Cell Biol. 11:33.

Kadler K. (1994) Extracellular matrix. 1: fibril-forming collagens. Protein Profile, 1(5):519-638. Review.

Schegg B, Hülsmeier AJ, Rutschmann C, Maag C, Hennet T. (2009) Core glycosylation of collagen is initiated by two beta(1-O)galactosyltransferases. Mol Cell Biol. 29(4):943-52.

https://doi.org/10.7554/eLife.41301.031

Article and author information

Author details

  1. Hieng Chiong Tie

    School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
    Contribution
    Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2738-8685
  2. Alexander Ludwig

    School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
    Contribution
    Resources, Formal analysis, Investigation, Visualization, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0696-5298
  3. Sara Sandin

    1. School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
    2. NTU Institute of Structural Biology, Nanyang Technological University, Singapore, Singapore
    Contribution
    Resources, Funding acquisition
    Competing interests
    No competing interests declared
  4. Lei Lu

    School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
    Contribution
    Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    lulei@ntu.edu.sg
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8192-1471

Funding

Ministry of Education - Singapore (Tier3 MOE2012-T3-1-001)

  • Sara Sandin

Ministry of Education - Singapore (Tier1 RG132/15)

  • Lei Lu

Ministry of Education - Singapore (Tier1 RG35/17)

  • Lei Lu

Ministry of Education - Singapore (Tier1 RG48/13)

  • Lei Lu

Ministry of Education - Singapore (Tier2 MOE2015-T2-2-073)

  • Lei Lu

National Medical Research Council (NMRC/CBRG/007/2012)

  • Lei Lu

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We would like to thank A Luini for discussions and the following persons for sharing their reagents with us: H Clausen, M De Matties, D Gadella, H Hauri, W Hong, A Linstedt, M Lowe, F Perez, J Rothman, E Snapp, Z Song, D Stephens, A Ting, F van Kuppeveld and Y Zhuang. This work was supported by the following grants to LL: NMRC/CBRG/007/2012, MOE AcRF Tier1 RG132/15, Tier1 RG35/17, Tier1 RG48/13 and Tier2 MOE2015-T2-2-073. This work was further supported (to SS) by the NTU Institute of Structural Biology (NISB) and a MOE Tier3 grant (MOE2012-T3-1-001).

Senior Editor

  1. Anna Akhmanova, Utrecht University, Netherlands

Reviewing Editor

  1. Suzanne R Pfeffer, Stanford University, United States

Publication history

  1. Received: August 21, 2018
  2. Accepted: November 30, 2018
  3. Accepted Manuscript published: November 30, 2018 (version 1)
  4. Version of Record published: December 14, 2018 (version 2)

Copyright

© 2018, Tie et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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