Living cells contain many membrane-bound compartments surrounded by a gel-like substance called the cytoplasm. Lysosomes are compartments found in most animal cells, which contain enzymes that can break down virtually all kinds of biological molecules. Cell biologists around the world use two proteins called LAMP1 and LAMP2 to mark lysosomes to study them. The loss of LAMP2 causes a condition called Danon disease that is characterized by thickening of the heart muscle. However, relatively little is known about what these proteins actually do.

Previous studies had hinted that these proteins might bind to the fatty molecule, cholesterol. Li and Pfeffer set out to test this directly and showed that LAMP1 and LAMP2 proteins do indeed bind to cholesterol. The two LAMP proteins also interact with another two proteins, called NPC1 and NPC2, which export cholesterol out of lysosomes.

Li and Pfeffer then showed that cells contain 5- to 10-times more LAMP proteins than they do NPC1-cholesterol exporters. This suggests that LAMP proteins have additional roles that need to be characterized and studied to see how important cholesterol binding is for these processes too. Future studies could also explore if LAMP proteins signal that free cholesterol is available for the cell’s needs.

Eukaryotic lysosomes are acidic, membrane-bound organelles that contain proteases, lipases and nucleases and degrade cellular components to regenerate catabolic precursors for cellular use (Xu and Ren, 2015; Schwake et al., 2013; Saftig and Klumperman, 2009). Lysosomes are crucial for the degradation of substrates from the cytoplasm, as well as membrane bound compartments derived from the secretory, endocytic, autophagic and phagocytic pathways. The limiting membrane of lysosomes is lined with so-called lysosomal membrane glycoproteins (LAMPs) that are comprised of a short cytoplasmic domain, a single transmembrane span, and a highly, N- and O-glycosylated lumenal domain (Wilke et al., 2012; Kundra and Kornfeld, 1999; Granger et al., 1990). Because of their abundance and glycan content, LAMPs have been proposed to serve as a protective barrier to block hydrolase access to the limiting phospholipid bilayer. LAMP1 and LAMP2 are 37% identical and may overlap in function, but knockout of LAMP1 in mouse has a much milder phenotype than depletion of LAMP2 (Tanaka et al., 2000): LAMP2-deficient mice have very short lifespans, and show massive accumulation of autophagic structures in most tissues. Indeed, LAMPs are required for fusion of lysosomes with phagosomes (Huynh et al., 2007) and LAMP2 has also been proposed to serve as a receptor for chaperone-mediated autophagy (Cuervo and Dice, 1996, 2000; Bandyopadhyay et al., 2008).

Previous work has implicated LAMP2 in cholesterol export from lysosomes, as LAMP-deficient cells show cholesterol accumulation that can be rescued by LAMP2 expression (Eskelinen et al., 2004; Schneede et al., 2011). Proteome-wide analysis of cholesterol binding proteins included LAMP1 and LAMP2 among a long list of candidate proteins (Hulce et al., 2013). Despite these hints, the precise function of LAMP proteins has remained unclear, and they are often presumed to be structural components. We show here that LAMP proteins bind cholesterol directly and this capacity contributes to their role in cholesterol export from lysosomes.

We sought to verify direct cholesterol binding to LAMP proteins using LAMP protein lumenal domains, engineered to be secreted from cells by simple deletion of their transmembrane and short cytoplasmic domains (Figure 1—figure supplement 1; Figure 1A). Soluble, purified, LAMP1 and LAMP2 proteins appeared to bind ³H-cholesterol saturably, at a stoichiometry comparable to equimolar amounts of purified, NPC1 N-terminal domain (NTD) that contains a single cholesterol binding site (Infante et al., 2008; Kwon et al., 2009; Figure 1B,D,E) (note that this does not provide information about relative binding affinities). Binding was not especially sensitive to pH (Figure 1H) and was complete after ~2 hr at 4°C (Figure 1I).

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Cholesterol is poorly soluble, thus binding reactions were carried out in the presence of sub-critical micelle concentration amounts of Nonidet P40 detergent (0.004%) to help solubilize the cholesterol, as worked out by Infante et al. in their studies of cholesterol binding to the N-terminal domain of NPC1 protein (Infante et al., 2008). Under these conditions, most of the cholesterol remains in a mixed micelle of cholesterol and detergent and is still poorly soluble. Thus, the apparent affinity for cholesterol is likely to be tighter than the curves indicate, as the amounts added do not reflect the concentration of free cholesterol that is actually available for binding.

Preliminary experiments showed that ³H-cholesterol binding was competed by unlabeled cholesterol, 24-hydroxycholesterol, but not cholesterol sulfate (Figure 1G). This suggested that binding occurs via the 3β-hydroxyl moiety of the cholesterol molecule, similar to the orientation with which the NPC1 N-terminal domain binds cholesterol (Kwon et al., 2009). Consistent with this, LAMP2 also bound ³H-25-hydroxycholesterol with similar apparent affinity as cholesterol; binding was competed by excess cold 25-hydroxycholesterol (Figure 1C), as would be expected for a specific interaction. Only low levels of background binding were detected using GFP-binding protein or TIP47 as controls (Figure 1D,E). More detailed analysis confirmed that 25-hydroxycholesterol (Figure 2B) and 7-ketocholesterol (Figure 2D), but not cholesterol sulfate (Figure 2A), compete with ³H-cholesterol for binding to LAMP2 protein.

Figure 2

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Epicholesterol is a cholesterol epimer that differs only in the chirality of carbon 3 such that the hydroxyl group is in the alpha rather than beta conformation. Importantly, epicholesterol failed to compete for cholesterol binding to LAMP2 protein under conditions where cholesterol competed for binding (Figure 2C). It was not possible to add higher concentrations of sterol competitors due to solubility issues, but significant inhibition was observed. Together, these data strongly support the conclusion that cholesterol binds LAMP2 via its 3β-hydroxyl moiety.

A slight stimulation of binding was seen in reactions containing low levels of competitor cholesterol sulfate or 25-hydroxycholesterol (Figure 2A,B); this is presumably due to the higher solubility of these sterols, which will help solubilize ³H-cholesterol present in the reaction’s mixed micelles, and presumably make it more available for LAMP2 binding (see also ref. Infante et al., 2008). Despite its somewhat higher solubility, 25-hydroxycholesterol did not appear to bind LAMP2 much more tightly than cholesterol, at least as inferred from its ability to compete with cholesterol for binding (Figure 2B) or to bind directly (Figure 1C).

The LAMP protein family includes LAMP1, LAMP2, DC-LAMP, BAD-LAMP and Macrosialin (Wilke et al., 2012). Each of these proteins contains a related 'LAMP' domain; LAMP1 and LAMP2 proteins each contain two (Figure 1—figure supplement 1). Soluble versions of the individual, membrane distal ('domain 1') and membrane proximal ('domain 2') LAMP domains of LAMP2 (Figure 1A) bound cholesterol with different capacity: the N-terminal, domain 1 bound more cholesterol than its membrane proximal, domain 2 counterpart under these conditions (Figure 1F). It is possible that both are capable of binding cholesterol within the full length molecule, as total domain 1 binding was less than that seen with the full length, secreted LAMP2 construct (Figure 1F).

To verify that LAMP2 binds cholesterol in cells, soluble LAMP2 protein was expressed and purified from the secretions of HEK293F cells grown in protein-free, FreeStyle 293 Expression Medium that does not contain cholesterol. Under these conditions, any LAMP2-bound sterol must come from intracellular sources. We subjected freshly purified LAMP2 protein to chloroform:methanol extraction and analyzed the extract by thin layer chromatography. As shown in Figure 3C, the LAMP2 extract contained a molecular species that co-chromatographed with cholesterol but not 24-hydroxy-, 25-hydroxy-, or 26-hydroxycholesterol, lanosterol or 7-beta-hydroxycholesterol. Mass spectrometry of the eluted material (Figure 3B) confirmed a profile identical with purified cholesterol standard (Figure 3A) (Figure 3C). These experiments show that LAMP2 purified from cell secretions carries primarily, bound cholesterol.

Figure 3

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NPC1 and NPC2 proteins mediate cholesterol export from lysosomes (Kwon et al., 2009; Rosenbaum and Maxfield, 2011). NPC1 has 13 transmembrane domains, and three large, lumenal domains that are important for its function. As mentioned earlier, the NPC1 N-terminal domain binds cholesterol directly (Kwon et al., 2009). Because of a possible connection between LAMP protein cholesterol binding and NPC-mediated cholesterol export, we checked for an interaction between these proteins. Membrane anchored, endogenous LAMP2 co-immuno-precipitated with full length NPC1-GFP but not with the control protein, GFP (Figure 4A) or the lysosomal membrane protein, MCOLN1 (Figure 4D), upon expression in HEK293T cells. Interestingly, co-immunoprecipitation decreased in cells treated for 24 hr with cyclodextrin to remove cholesterol from lysosomes (Abi-Mosleh et al., 2009; Rosenbaum et al., 2010) and the plasma membrane (Figure 4B,C). These conditions (~0.1% cyclodextrin) have been shown to be non-toxic (cf. Ulloth et al., 2007) and did not alter cell growth rate or viability in our hands.

Figure 4

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Purified, soluble LAMP2 protein also bound very tightly (and directly) to the N-terminal domain of NPC1 protein (Figure 5A, red) in the presence (or absence, not shown) of cholesterol (K_D = 6 nM), as monitored by microscale thermophoresis using AF647 dye-conjugated NPC1 protein—binding significantly altered the fluorescence of NPC1 protein (Figure 5C). No interaction was observed for the control glycoprotein, RNase B (Figure 5E). The smaller, NPC2 protein (Figure 5B) also bound to LAMP2 (K_D = 122 nM), but ~20 fold less tightly that NPC1 N-terminal domain (Figure 5D); binding was monitored in the presence of cholesterol sulfate which will occupy the binding site of NPC2 but not LAMP2 (Figures 1 and 2). No binding was seen for NPC2 to the control RNase B protein (Figure 5F). Thus, LAMP2 binds directly to both NPC1 N-terminal domain and NPC2 proteins. For NPC1, the enhanced binding seen in cells in the presence of cholesterol does not appear to reflect occupancy of NPC1’s N-terminal domain, as this variable did not influence LAMP2 binding in solution. However, it is important to note that NPC1 also likely binds cholesterol within its membrane spanning region which may also influence its overall conformation (Lu et al., 2015; Li et al., 2016).

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LAMP proteins are highly abundant components of the lysosome membrane and may serve as a reservoir for cholesterol extracted from intra-lysosomal membranes by NPC2, prior to cholesterol export from lysosomes by NPC1. [The term, reservoir, is meant to imply a holding station for cholesterol molecules that have been solubilized from the internal lipid contents of lysosomes by NPC2, and held closer to the limiting membrane, prior to NPC1-mediated export.]

Are LAMP proteins abundant enough to represent a cholesterol reservoir? We used purified LAMP2 and NPC1 proteins as standards to determine their precise abundance in HeLa and HEK293 cell lysates (Figure 5—figure supplement 1). Using the polypeptide molecular weights and cellular protein determinations, we estimate that HeLa cells contain 6.8 × 10⁶ LAMP2 molecules per cell and 3.7 × 10⁵ NPC1 molecules per cell, or 18 fold more LAMP2 than NPC1; HEK293T cells contain 2 × 10⁶ LAMP2 molcules per cell and 5.9 × 10⁵ NPC1 molecules (3.6 × fold more LAMP2).

Baby hamster kidney cells have been estimated to contain an absolute volume of ~37 µm³ lysosomes and prelysosomes per cell (3.7 × 10^–14 l) and a lysosome membrane area of 370 µm² (Griffiths et al., 1989). Assuming similar values for HeLa cells, this would represent a LAMP2 membrane density of 18,378 or 5676 molecules per µm² in HeLa or 293T cell lysosomes, respectively, consistent with previous reports (Granger et al., 1990). For comparison, tightly packed viral spike glycoproteins occur at a density of 22,000 molecules per µm² (Quinn et al., 1984). We assume that LAMP1 will be of similar high density, together with LAMP2, practically lining the interior of the lysosome limiting membrane. In terms of concentrations, 37 femtoliters of lysosome volume would contain 0.3 mM LAMP2-associated cholesterol binding sites in HeLa cell lysosomes (assuming one mole cholesterol bound per more LAMP2). It does not seem unreasonable to consider this as a significant reservoir of cholesterol molecules that may be poised for transfer to NPC1 protein prior to export.

Residues needed for cholesterol binding are needed for LAMP protein function

The structure of an individual LAMP domain from DC-LAMP protein is comprised of a novel, beta-prism fold that appears to contain a hydrophobic pocket (Wilke et al., 2012); we used this structure to model the structure of LAMP2 domain 1 (Figure 6A). Site directed mutagenesis of hydrophobic residues predicted to line the walls of this cavity yielded purified LAMP2 proteins with impaired cholesterol binding activity. Thus, a soluble, LAMP2 domain 1-I¹¹¹A/V¹¹⁴A construct yielded a secreted protein (Figure 6B inset, right lane) that bound significantly less cholesterol than its wild type counterpart (Figure 6B inset, left lane and panel B). Because these proteins were obtained from cell secretions, they are likely to be properly folded, as they escaped the endoplasmic reticulum’s quality control machinery. These experiments show that residues facing the predicted, prism fold pocket are important for cholesterol binding and likely contribute to the cholesterol binding site.

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Finally, to verify the importance of cholesterol binding to LAMP2 protein as part of its physiological role, we tested the ability of wild type and mutant LAMP2 constructs to rescue the cholesterol accumulation seen in lysosomes from mouse embryonic fibroblasts missing LAMP1 and LAMP2 proteins (Eskelinen et al., 2004; Schneede et al., 2011). The ability of lysosomes to export cholesterol can be monitored by feeding cells cholesterol in the form of LDL, and using conversion of ¹⁴C-oleic acid to cholesteryl oleate that takes place after endocytosed cholesterol is transported to the endoplasmic reticulum (Goldstein et al., 1983). Previous work showed that LAMP1/2 knockout MEF cells were impaired in cholesterol export using this assay (Schneede et al., 2011).

We used lentivirus transduction to test the ability of full length LAMP2, a membrane anchored LAMP2 domain 1 (LAMP2-GFP Δ194–368; Figure 1—figure supplement 1), or a membrane anchored LAMP2 domain 1-I¹¹¹A/V¹¹⁴A to rescue the ability of LAMP1/LAMP2 knockout MEF cells to export LDL-derived cholesterol from lysosomes. For these experiments, we used LAMP constructs containing a single LAMP domain, as full length LAMP2 constructs with mutations in both LAMP domains failed to fold properly or be transported efficiently to lysosomes.

It was important to first verify the precise amounts of each construct in lysosomes, to evaluate any functional rescue findings. Flow cytometry analysis showed that the rescue constructs were expressed at comparable levels in each stably expressing cell population (Figure 6C). Light microscopy confirmed that the constructs were capable of proper lysosome localization, as determined by their colocalization with endogenous LAMP1 protein (Figure 6E) in HeLa cells. (Similar staining was observed in LAMP knockout MEF cells that lack LAMP protein markers).

To fully confirm the folding of these artificial constructs, we analyzed their glycosylation status and stability after addition of cycloheximide to inhibit new protein synthesis (Figure 6—figure supplement 1). Full length GFP-LAMP2 protein migrated at ~140 kD and its abundance was not altered after 4 hr cycloheximide treatment, consistent with its long half life in cultured cells (panel B). Similarly, the GFP-domain 1 construct was stable under these conditions and migrated at ~90 kD (panels A,B). In contrast, the I¹¹¹A/V¹¹⁴A mutant domain I protein displayed two distinct bands; the upper band was stable, while the lower band likely corresponded to an ER form that was largely degraded after 4 hr in cycloheximide (panels A,B). From this we conclude that cells expressing membrane anchored LAMP2 domain 1 I¹¹¹A/V¹¹⁴A are less efficient at folding the protein but some folded protein makes it to lysosomes, where it is stable. This difference was accounted for in subsequent functional rescue experiments (Figure 6D).

Figure 6D shows that as expected, full length, wild type LAMP2 rescued cholesterol export in LAMP1/2-deficient MEF cells; membrane anchored LAMP2 domain 1 showed a level of rescue consistent with its lower capacity for cholesterol binding (cf. Figure 1F). Importantly, membrane anchored, LAMP2 domain 1 I¹¹¹A/V¹¹⁴A failed to rescue cholesterol export from lysosomes (Figure 6D), consistent with its inability to bind cholesterol; shown are the data corrected for the amount of mature proteins present in lysosomes in these cells. LAMP2 constructs mutated in both cholesterol-binding sites could not be tested, as they were only poorly delivered to lysosomes.

These experiments demonstrate a direct role for LAMP2 in cholesterol export from lysosomes, and confirm that LAMP2’s ability to bind cholesterol correlates with its ability to support cholesterol export from LAMP-deficient MEF cells. In addition, LAMP proteins bind tightly to NPC proteins in vitro and in cells, and appear to facilitate cholesterol export from lysosomes.

LAMP proteins are the most highly abundant membrane glycoproteins of the lysosome, and their lumenally oriented cholesterol binding sites represent a significant binding site for this important sterol. We measured ~7 × 10⁶ molcules per HeLa cell, representing 0.3 mM binding sites in lysosomes. A recent cellular mass spectrometry analysis (Itzhak et al., 2016) estimated LAMP proteins to be present at 260,000 copies and NPC1 at 29,193 copies per HeLa cell. While the relative abundance of these proteins matches the values we report here, their total level was 25 fold lower in that study. It is possible that these transmembrane glycoproteins were under-represented in due to their unusual protease resistance as proteins of the lysosome membrane, differences in cell confluency and/or differences in HeLa cell lines employed.

Lysosomes have recently been shown to sense and signal amino acid availability to influence lysosome biogenesis in relation to cellular need (Settembre et al., 2013), and LAMP oligomerization has been reported to correlate with chaperone mediated autophagy (Bandyopadhyay et al., 2008). Cholesterol levels may influence LAMP protein conformation or interaction with other partners to signal the availability of endocytosed cholesterol to influence autophagy and cellular metabolism. The ten fold higher abundance of LAMP proteins compared with NPC1 protein in HeLa cells suggests that LAMP proteins may do more than just facilitate NPC1 function in cholesterol export. Future experiments will be needed to fully understand the roles played by these highly abundant lysosomal membrane glycoproteins.

We have shown that LAMP2 binds tightly to the N-terminal domain of NPC1 and also binds cholesterol with the same orientation as that domain. LAMP2 also aids in cholesterol export from lysosomes. How might LAMP2’s cholesterol binding site contribute to cholesterol export? Current models suggest that the soluble NPC2 protein binds cholesterol from the internal membranes of lysosomes and delivers it to NPC1 at the limiting membrane of this compartment (Kwon et al., 2009). One possibility is that NPC2 can deliver cholesterol to both NPC1 and to LAMP2, which is more abundant. This would help drive the cholesterol export process by moving cholesterol from the accumulated, lumenal lipid stores to the lysosome’s limiting membrane. Because LAMP2 and NPC1 N-terminal domains bind cholesterol in the same orientation, it makes sense that NPC2 (which binds in opposite orientation, [Xu et al., 2007]) could transfer the cholesterol between these two proteins. The recent crystal structure of NPC2 bound to the middle, lumenal domain of NPC1 (Li et al., 2016) supports a direct handoff between NPC1 and NPC2 (Kwon et al., 2009). In future work, it will be important to elucidate precisely how LAMP2 interacts with both NPC2 and NPC1 to facilitate cholesterol export from lysosomes and how cholesterol binding contributes to LAMP2’s other cellular roles.

Cholesterol, epicholesterol and sodium cholesteryl sulfate were from Sigma (St. Louis, MO); 24-hydroxycholesterol (24-HC) was a gift from Rajat Rohatgi (Stanford University, Stanford, CA); 25-hydroxycholesterol and 7-ketocholesterol were from Steraloids (Newport, RI) or Avanti Polar Lipids (Alabaster, AL); [1,2-³H]cholesterol (50 Ci/mmol) and 25-[26,27-³H] hydroxycholesterol were from American Radiolabeled Chemicals (St. Louis, MO). Ni-NTA agarose was from Qiagen (Valencia, CA); freestyle 293 expression medium and Dulbecco’s modified Eagle’s medium (DMEM) was from Life Technologies (Carlsbad, CA); lipoprotein deficient serum was from KALEN Biomedical (Montgomery Village, Maryland). Pierce Protein Concentrators PES were from Thermo Fisher Scientific (Grand Island, NY); PD-10 desalting columns and Q-Sepharose were from GE Healthcare Life Sciences (Pittsburgh, PA); pFastBac NPC1-N-terminal domain plasmid, LAMP1-mGFP and MCOLN1-pEGFP C3 were from Addgene (Cambridge, MA); pGEM-LAMP2 was from Sino Biological Inc; mouse anti-human LAMP1 and LAMP2 antibody culture supernatants were from Developmental Studies Hybridoma Bank (University of Iowa, Iowa city, IA). Rabbit monoclonal anti-NPC1 was from AbCam (Cambridge, MA); Chicken anti-GFP antibody was from Aves Labs (Tigard, Oregon); IRDye 800CW donkey anti-chicken and IRDye 680RD donkey anti-mouse antibodies were from LI-COR, Inc. (Lincoln, NE); anti-chicken-HRP conjugate was from Promega (Sunnyvale, CA); goat anti-mouse-HRP conjugate was from BioRad (Hercules, CA); ECL Western Blotting Substrate was from Thermo Scientific (Rockford, IL).

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Author details

1. Jian Li

   Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

   Contribution

   JL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

2. Suzanne R Pfeffer

   Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

   Contribution

   SRP, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   pfeffer@stanford.edu

   Competing interests

   SRP: Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0002-6462-984X

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