A microscopy-based kinetic analysis of yeast vacuolar protein sorting
- Department of Molecular Genetics and Cell Biology, University of Chicago, United States
Figures
Figure 1
A regulatable vacuolar cargo.
(A) General strategy for the use of reversibly aggregating fluorescent cargoes. DsRed-Express2 tetramers (red) are linked to a dimerizing FKBP variant (gold), so the tetramers associate to form …
Figure 2 with 2 supplements
Traffic kinetics of the vacuolar cargo.
(A) Visualizing cargo traffic. The vacuolar cargo expressed in VPS10 wild-type or vps10∆ strains was imaged by 4D confocal microscopy. Prior to the video, fluorescence from leaked cargo molecules …
Figure 2—figure supplement 1
Minor effect of cycloheximide treatment on the kinetics of cargo traffic to the vacuole.
(A) Comparison of traffic kinetics in the absence or presence of cycloheximide (CHX). Data from Figure 2C were re-plotted with the additional analysis of VPS10 cells that had been pretreated with …
Figure 2—video 1
Visualizing traffic of the vacuolar cargo from the ER to the vacuole.
The cargo expressed in VPS10 wild-type (WT) and vps10∆ strains was imaged by 4D confocal microscopy together with the vacuolar membrane marker Vph1-GFP. Prior to the video, fluorescence from leaked …
Figure 3 with 2 supplements
Sequential appearance of the vacuolar cargo in Golgi and PVE compartments.
(A) Appearance of the vacuolar cargo in early Golgi compartments marked with GFP-Vrg4 and in late Golgi compartments marked with Sec7-HaloTag. Cells were grown to mid-log phase, labeled with JF646, …
Figure 3—video 1
Visualizing traffic of the vacuolar cargo together with Golgi markers.
Cells expressing the cargo together with the early Golgi marker GFP-Vrg4 and the late Golgi marker Sec7-HaloTag were grown to mid-log phase, labeled with JF646, and imaged by 4D confocal microscopy. …
Figure 3—video 2
Visualizing traffic of the vacuolar cargo together with PVE and vacuole markers.
Cells expressing the cargo together with the PVE marker Vps8-GFP and the vacuolar membrane marker Vph1-HaloTag were grown to mid-log phase, labeled with JF646, and imaged by 4D confocal microscopy. …
Figure 4 with 3 supplements
Visualizing the vacuolar cargo during Golgi maturation.
(A) Visualizing the vacuolar cargo in a VPS10 wild-type strain. Cells expressing the vacuolar cargo together with the early Golgi marker GFP-Vrg4 and the late Golgi marker Sec7-HaloTag were grown to …
Figure 4—figure supplement 1
Additional examples of vacuolar cargo traffic during Golgi maturation.
(A) Vacuolar cargo traffic in a VPS10 wild-type strain. The experiment was performed as in Figure 4A. Shown are average projected Z-stacks at representative time points from an additional video. The …
Figure 4—video 1
Visualizing traffic of the vacuolar cargo during Golgi maturation.
Cells expressing the cargo together with the early Golgi marker GFP-Vrg4 and the late Golgi marker Sec7-HaloTag were grown to mid-log phase, labeled with JF646, and imaged by 4D confocal microscopy. …
Figure 4—video 2
Visualizing traffic of the vacuolar cargo during Golgi maturation in a strain lacking Vps10.
The procedure was as in Figure 4—video 1 except that a vps10∆ strain was used. Frames from this video are shown in Figure 4D. Scale bar, 2 µm.
Figure 5 with 3 supplements
Kinetics of GGA arrival at the Golgi.
(A) Visualizing the dynamics of the GGA and AP-1 adaptors during cisternal maturation. A strain expressing the GGA protein Gga2-HaloTag, the AP-1 subunit Apl2-GFP, and the late Golgi marker …
Figure 5—figure supplement 1
Additional examples of adaptor dynamics and of the relationship between GGA arrival and vacuolar cargo departure.
(A) Visualizing the dynamics of the GGA and AP-1 adaptors during cisternal maturation. The experiment was performed as in Figure 5A. Shown are average projected Z-stacks at representative time …
Figure 5—video 1
Visualizing the dynamics of the GGA and AP-1 clathrin adaptors during Golgi maturation.
Cells expressing the GGA protein Gga2-HaloTag, the AP-1 subunit Apl2-GFP, and the late Golgi marker Sec7-mScarlet were grown to mid-log phase, labeled with JF646, and imaged by 4D confocal …
Figure 5—video 2
Visualizing the vacuolar cargo together with a GGA adaptor.
Cells expressing the cargo together with the early Golgi marker GFP-Vrg4 and the GGA protein Gga2-HaloTag were grown to mid-log phase, labeled with JF646, and imaged by 4D confocal microscopy. SLF …
Figure 6 with 4 supplements
Requirement for the GGAs but not AP-1 during Golgi-to-PVE traffic.
(A) Visualizing vacuolar cargo traffic during Golgi maturation in a strain lacking AP-1. The experiment was performed as in Figure 4A, except that an apl4∆ strain was used. Shown are average …
Figure 6—figure supplement 1
Additional examples of cargo dynamics during cisternal maturation in strains lacking AP-1 or GGAs.
(A) Visualizing vacuolar cargo traffic during Golgi maturation in a strain lacking AP-1. The experiment was performed with an apl4∆ strain as in Figure 6A. Shown are average projected Z-stacks at …
Figure 6—figure supplement 2
Secretion of the vacuolar cargo in cells lacking either Vps10 or GGAs.
(A) Immunoblot of secreted cargoes after SLF addition in rich medium. Wild-type (WT) cells expressing the secretory cargo and wild-type, vps10∆, apl4∆, and gga1∆ gga2∆ cells expressing the vacuolar …
Figure 6—video 1
Visualizing traffic of the vacuolar cargo during Golgi maturation in a strain lacking AP-1.
The procedure was as in Figure 4—video 1 except that an apl4∆ strain was used. Frames from this video are shown in Figure 6A. Scale bar, 2 µm.
Figure 6—video 2
Visualizing traffic of the vacuolar cargo during Golgi maturation in a strain lacking GGAs.
The procedure was as in Figure 4—video 1 except that a gga1∆ gga2∆ strain was used. Frames from this video are shown in Figure 6D. Scale bar, 2 µm.
Figure 7 with 2 supplements
Visualizing transfer of the vacuolar cargo from PVE compartments to the vacuole.
(A) Gradual movement of the vacuolar cargo from a PVE compartment to the vacuole. A strain expressing the vacuolar membrane marker Vph1-HaloTag, the PVE marker Vps8-GFP, and the vacuolar cargo was …
Figure 7—video 1
Visualizing movement of the vacuolar cargo from a PVE compartment to the vacuole.
Cells expressing the cargo together with the vacuolar membrane marker Vph1-HaloTag and the PVE marker Vps8-GFP were grown to mid-log phase, attached to a confocal dish, and treated with SLF for …
Figure 7—video 2
Visualizing sudden movement of the vacuolar cargo from a PVE compartment to the vacuole.
The procedure was as in Figure 7—video 1. Frames from this video are shown in Figure 7C. Scale bar, 2 µm.
Figure 8 with 5 supplements
Visualizing transfer of Mup1 from PVE compartments to the vacuole.
(A) Movement of Mup1 from a PVE compartment to the vacuole. A strain expressing the vacuolar membrane marker Vph1-HaloTag, the PVE marker Vps8-GFP, and Mup1-mScarlet was grown to mid-log phase in …
Figure 8—figure supplement 1
Reduction in Vps8 labeling of a PVE compartment after a large cargo transfer event.
(A) Sudden movement of Mup1 from a PVE compartment to the vacuole. The experiment was performed as in Figure 8A, and frames are shown from Figure 8—video 3. Orange arrows indicate the PVE …
Figure 8—figure supplement 2
Evidence from previously published electron tomography data (McNatt et al., 2007) for partial fusion of PVE compartments with the vacuole.
This image shows electron tomography of vacuoles and associated PVE compartments in S. cerevisiae. Non-fused PVE compartments are yellow, vacuoles are red, and PVE compartments with tubular …
Figure 8—video 1
Visualizing movement of Mup1 from a PVE compartment to the vacuole.
Cells expressing the vacuolar membrane marker Vph1-HaloTag, the PVE marker Vps8-GFP, and Mup1-mScarlet were grown to mid-log phase in NSD lacking methionine, attached to a confocal dish, and exposed …
Figure 8—video 2
Visualizing sudden movement of Mup1 from a PVE compartment to the vacuole.
The procedure was as in Figure 8—video 1. Frames from this video are shown in Figure 8C. Scale bar, 2 µm.
Figure 8—video 3
Reduction in the apparent size of a PVE compartment after a large cargo transfer event.
The procedure was as in Figure 8—video 1. Frames from this video are shown in Figure 8—figure supplement 1A. Scale bar, 2 µm.
Figure 9
Model for sorting of biosynthetic cargoes in the late Golgi.
(A) Sequential formation of GGA vesicles and AP-1 vesicles in yeast cells. The thick arrows represent progressive maturation of a Golgi cisterna over time. During the early-to-late Golgi transition …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Chemical compound, drug | Hygromycin | Thermo Fisher | Cat. #: 10687010 | |
| Chemical compound, drug | G418 | Teknova | Cat. #: G5001 | |
| Chemical compound, drug | Nourseothricin | Neta Scientific | Cat. #: RPI-N51200-1.0 | |
| Chemical compound, drug | JF646 HaloTag Ligand | Dr. Luke Lavis (Janelia Research Campus) Grimm et al., 2015 | ||
| Chemical compound, drug | SLF | Cayman Chemical | Cat. #: 10007974–5 | |
| Chemical compound, drug | Cycloheximide | Neta Scientific | Cat. #: RPI-C81040-1.0 | |
| Chemical compound, drug | Concanavalin A | Sigma-Aldrich | Cat. #: C2010-250MG | |
| Antibody | anti-FKBP12 (rabbit polyclonal) | Abcam | Cat. #: ab2918 | WB (1:1000) |
| Antibody | Alexa Fluor 647 anti-rabbit (goat polyclonal) | Thermo Fisher | Cat. #: A21245 | WB (1:1000) |
| Software, algorithm | Graphpad Prism | Insightful Science (https://www.graphpad.com) | RRID:SCR_002798 | |
| Software, algorithm | SnapGene | Insightful Science (https://www.snapgene.com) | RRID:SCR_015052 | |
| Software, algorithm | ImageJ | ImageJ (https://imagej.nih.gov/ij/) | RRID:SCR_003070 |
Table 1
Primers used in this study.
| Purpose | Amplifies | Primers |
|---|---|---|
| PDR1 deletion | kanMX resistance cassette | 5’-CAGCCAAGAATATACAGAAAAGAATCCAAGAAACTGGAAGCGTACGCTGCAGGTCGAC-3’ 5’-GGAAGTTTTTGAGAACTTTTATCTATACAAACGTATACGTATCGATGAATTCGAGCTCG-3’ |
| PDR3 deletion | Nourseothricin resistance cassette | 5’-ATCAGCAGTTTTATTAATTTTTTCTTATTGCGTGACCGCACGTACGCTGCAGGTCGAC-3’ 5’-TACTATGGTTATGCTCTGCTTCCCTATTTTCTTTGCGTTTATCGATGAATTCGAGCTCG-3’ |
| GGA1 deletion | Hygromycin resistance cassette | 5’-AGTCACTACTTCAAGTATAACCCAGACAAGAGTCTTTTAAATAGCTTGCCTTGTCCCCGC-3’ 5’-ATGGCATCTACTTTTTTTTCAACTTCTCTACCGAATTTGACGTTTTCGACACTGGATGGC-3’ |
| VPS10 deletion | VPS10 5’ upstream | 5’-CCCAAACTAAAAAGTATCCGCCTGT-3’ 5’-GACAAGGCAAGCTAACGTGTGATGACTACTGGACACT-3’ |
| VPS10 3’ downstream | 5’-GCCATCCAGTGTCGAAGAGATTACTTTACATAGAGTAGATAATTCCATATACTTTTCATA −3’ 5’-AATGAAGTACTATAAATATTAAAGTACGTTAGTAGTTTATTTCTCTTCGG-3’ | |
| Hygromycin resistance cassette | 5’-TCATCACACGTTAGCTTGCCTTGTCCCCGC-3’ 5’-TGTAAAGTAATCTCTTCGACACTGGATGGCGG-3’ | |
| APM3 deletion | APM3 5’ upstream | 5’-AGGGGTAGAAGTCGCTGATTGAT-3’ 5’-GGGCCTCCATGTCCTATTTTGGTTGGGTTGGTAAGGTTTACAG-3’ |
| APM3 3’ downstream | 5’-GCTGGTCGCTATACTGTTATATGTGTACTTGAAATTCCATGCGAAACTAAA-3’ 5’-TGCGGAAGTCTTCCCTAAGACG-3’ | |
| Hygromycin resistance cassette | 5’-CAACCAAAATAGGACATGGAGGCCCAGAATACCC-3’ 5’-TCAAGTACACATATAACAGTATAGCGACCAGCATTCACA-3’ | |
| APL4 deletion | APL4 5’ upstream | 5’-ATGTATATAATTCCGGAAGTGTGGTCCT-3’ 5’-GACAAGGCAAGCTTATGGTGTTCAGGTCTTTCTCGTTGCT-3’ |
| APL4 3’ downstream | 5’-CCATCCAGTGTCGAAAAATGCCTTTAAAATTACAGAACATAACATGATTAATGAC-3’ 5’-GAATTCTGGTCCAAGGCAATTCTATATTTGAT-3’ | |
| Hygromycin resistance cassette | 5’-CCTGAACACCATAAGCTTGCCTTGTCCCCG-3’ 5’-TTTTAAAGGCATTTTTCGACACTGGATGGCGG-3’ | |
| GGA2 deletion | GGA2 5’ upstream | 5’-GATTTCTACAGTCTTTCTGATGGGTTCTTGG-3’ 5’-ACGATATTCTTAGACATGATGCAGTATCACGATTAGCAAT-3’ |
| GGA2 3’ downstream | 5’-AATCTTGGCTTAATCCTCTGGCGTTTCTTATCAATCCTTTCT-3’ 5’-TCTTCCTTTGAAGAAAATTCGTCCTCATCT-3’ | |
| K. lactis LEU2 | 5’-AATCGTGATACTGCATCATGTCTAAGAATATCGTTGTCCTACCGG-3’ 5’-GAAACGCCAGAGGATTAAGCCAAGATTTCCTTGACAGCC-3’ | |
| Integration at TRP1 | TRP1 locus | 5’-GTGTACTTTGCAGTTATGACG-3’ 5’-AGTCAACCCCCTGCGATGTATATTTTCCTG-3’ |
Additional files
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Supplementary file 1
Plasmid files.
- https://cdn.elifesciences.org/articles/56844/elife-56844-supp1-v2.zip
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Transparent reporting form
- https://cdn.elifesciences.org/articles/56844/elife-56844-transrepform-v2.docx
