Differential Effects of TBC1D15 and Mammalian Vps39 on Rab7 Activation State, Lysosomal Morphology, and Growth Factor Dependence^*

Antibodies, Plasmids, and Reagents

Rab7 antibody (clone Rab7-117) was obtained from Sigma. Human Lamp1 antibody (clone eBioH4A3) was obtained from eBioscience. The Myc antibody (clone 9B11) was obtained from Cell Signaling. The eGFP antibody (clone JL-8) was obtained from Clontech. Secondary antibodies were obtained from LI-COR Biosciences. Complete protease inhibitors were from Roche Diagnostics. BCA reagents used for protein assays were from Pierce Chemical Co. Murine TBC1D15 was amplified by PCR from pSport1 IMAGE clone 30054233 and an N-terminal Myc tag added. The PCR product was TA subcloned into pEF6/V5-His (Invitrogen), sequence confirmed, and moved into the retroviral vector pBABEpuro. Human GFP-DN-RILP and GFP-RILP were generously provided by Cecilia Bucci (Università del Salento, Lecce, Italy). To generate an mVps39 expression construct, RNA was isolated from FL5.12 cells using a Qiagen RNAeasy kit. The mVps39 cDNA was generated using a Superscript III RT-PCR kit with Platinum Taq (Invitrogen) and TA cloned into the EF6/V5-His vector (Invitrogen). An N-terminal Myc tag was added during RT-PCR. Sequencing of the entire insert identified four mutations in mVps39; this construct was renamed mVps39_mut. Wild-type mVps39 was kindly provided by Dr. Robert Piper (University of Iowa) and cloned into pBABEpuro. GFP-Rab5-S34N was generously provided by Philip Stahl (Washington University School of Medicine). GFP-Rab11-S25N was obtained from Richard Pagano via Addgene (Plasmid 12678). All plasmids were sequence confirmed.

Cell Culture and Transfection

FL5.12 cells were maintained at low density in RPMI (Mediatech) supplemented with 10% FCS (Mediatech, Hyclone, or Sigma), 500 pm recombinant mouse IL-3 (BD Pharmingen or eBioscience), 10 mm HEPES (Mediatech), 55 μm β-mercaptoethanol (Sigma), antibiotics, and l-glutamine (Mediatech). Cell lines expressing transgenes were used in experiments within 5 days of thawing. Extracellular nutrient limitation was performed in RPMI made from chemical components that contained 10% dialyzed FCS (Invitrogen) in place of standard serum. Hela and 293T cells were maintained in Dulbecco's modified Eagle's medium (Mediatech) supplemented with 10% FCS. Transfections were performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

GST-RILP Pull-downs

Nucleotides 658–897 of GenBank^TM accession number NM 001029938 (amino acids 220–299) of the murine RILP protein constituting the Rab7 binding domain of RILP were fused to the C terminus of GST in the pGEX 4T-3 vector (Stratagene). GST-RILP was transformed into Escherichia coli strain BL21. 250 ml of LB was inoculated with 1 ml of an overnight culture and grown at 37 °C to an OD of 0.6 to 0.8. Isopropyl-1-thio-β-d-galactopyranoside (EMD) was then added to a final concentration of 0.5 mm to induce protein production. The 250-ml culture was incubated for additional 3–4 h at 30 °C, after which the bacteria were spun down, washed with cold PBS, resuspended in 5 ml of cold lysis buffer (25 mm Tris-HCl pH 7.4, 1 m NaCl, 0.5 mm EDTA, 1 mm dithiothreitol, 0.1% Triton X-100, with Complete protease inhibitors), then sonicated. The bacterial lysates were cleared by centrifugation, and 5 ml of cold lysis buffer was added. Proteins were purified by adding 300 μl of a pre-equilibrated 50% slurry of glutathione-Sepharose 4B beads (GE Healthcare) to the lysate. Beads were incubated with lysates for 30 min. at room temperature then washed with lysis buffer, resuspended as a 50% slurry, and protein levels quantified using the BCA Assay. Mammalian cells to be analyzed in the pull-down were lysed in pull-down buffer (20 mm HEPES, 100 mm NaCl, 5 mm MgCl₂, 1% Triton X-100, protease inhibitors). Each pull-down was performed in 1 ml with 300 μg of cell lysate and 30 μg of beads pre-equilibrated in pull-down buffer. Beads were rocked overnight at 4 °C, washed twice with cold pull-down buffer, and bound proteins eluted by adding 2× NuPAGE sample buffer (Invitrogen) and incubating at 72 °C for 10 min.

Flow Cytometry and Microscopy

Cells were analyzed on a Becton Dickinson LSR II flow cytometer. Viability was determined by vital dye exclusion (propidium iodide or DAPI, Invitrogen). To evaluate lysosomal morphology, cells were stained with 500 nm Lysotracker Red (Invitrogen) for 30 min. at 37 °C and examined using a Nikon Eclipse TE2000 fluorescence microscope equipped with a Coolsnap CCD camera. Quantitation of the number of lysosomes was performed manually using the tagging function available in ImagePro software. To evaluate Lamp1 clustering, transfected Hela cells growing on coverslips were fixed and permeabilized in IF block (PBS with 10% FCS, 2% paraformaldehyde, 0.3% saponin, and 0.05% sodium azide) for 15 min at room temperature. Cells were washed twice in IF wash (PBS with 0.03% saponin) before and after staining with antibodies.

Transferrin Internalization and Recycling

FL5.12 cells were washed with PBS and resuspended in serum-free medium with 1% bovine serum albumin. The cells were incubated at 37 °C for 1 h then washed with ice-cold PBS before labeling on ice with 100 μg/ml biotinylated transferrin (Invitrogen) in serum-free medium with 1% BSA. Cells were washed with ice-cold PBS to remove unbound transferrin and incubated at 37 °C in complete medium with 20% FCS. At the indicated time points, cells were washed in ice-cold PBS with 2% fetal bovine serum and 0.05% sodium azide, fixed with 1% paraformaldehyde (on ice for 30 min. followed by 10 min at room temperature), stained with streptavidin-APC (eBioscience), and analyzed on a BD LSRII flow cytometer. For transferrin recycling assays, cells were labeled with biotinylated transferrin at 37 °C for 1 h before transfer to complete medium containing 20% FCS and permeabilized with saponin during fixation. Where indicated, Rab5 and Rab11 mutant plasmids were introduced by electroporation.

TBC1D15 Is a Rab7-selective GAP

TBC1D15 can function as a GAP for Rab7 in vitro (39). However, GAPs are often promiscuous in vitro while displaying selective activity in a cellular context. To test whether TBC1D15 serves as a Rab7 GAP in cells, we developed an effector pull-down assay in which the Rab7-binding domain of its effector protein RILP (49) was fused to GST and used to selectively isolate Rab7-GTP from cell lysates. Pull-down assays on cells expressing Rab7 mutants that are predominantly GDP- (Rab7-T22N) or GTP-bound (Rab7-Q67L) confirmed that Rab7-GTP was selectively isolated by GST-RILP (Fig. 1, A and B) (2, 13, 15). To further validate this assay, we evaluated Rab7-GTP binding status in cells expressing mutant or wild-type RILP. In keeping with published results (13, 15), both DN-RILP and wild-type RILP increased Rab7-GTP levels (Fig. 1, C and D). The SifA protein from Salmonella enterica interferes with RILP recruitment to the Salmonella containing vacuole, possibly by serving as a competitive inhibitor (45). Unlike DN-RILP, SifA does not alter the Rab7 GTPase cycle (50). As expected, no change in Rab7-GTP levels was observed in cells expressing SifA (Fig. 1, C and D). These experiments demonstrate that the effector pull-down assay has the dynamic range to detect both reductions and elevations in Rab7-GTP binding.

Consistent with the ability of TBC1D15 to function as a Rab7 GAP in vitro (39), the fraction of Rab7 bound to GTP was reduced in cells expressing TBC1D15 (Fig. 1, C and D). As a functional correlate, we tested whether TBC1D15 overexpression altered lysosomal morphology. TBC1D15 expression led to lysosomal fragmentation (Fig. 2, A and B). The degree of fragmentation was similar to that observed upon expression of the dominant-negative mutant Rab7-T22N. Fragmentation induced by TBC1D15 was not reversed by expression of Rab7-Q67L, possibly because high enough levels of the activated mutant were not achieved. In addition to fragmenting the lysosome, inactivating Rab7 decreases cellular sensitivity to growth factor withdrawal-induced apoptosis (4). Cells stably overexpressing TBC1D15 were also protected from growth factor withdrawal-induced death (Fig. 2C). Together, these results are consistent with a role for TBC1D15 as a Rab7 GAP.

TBC1D15 can function as a Rab11 GAP in vitro (39) and an association between TBC1D15 and Rab5 was detected in yeast two-hybrid assays (51). To determine whether TBC1D15 might also serve as a GAP for these Rabs, we measured transferrin endocytosis and recycling in cells overexpressing TBC1D15. Transferrin internalization depends on the function of both Rab4 and Rab5 while Rab4 and Rab11 promote transferrin recycling (3, 52–58). Both transferrin internalization and recycling are, on the other hand, independent of Rab7 (3, 59). To measure transferrin internalization, biotinylated transferrin was bound to FL5.12 cells on ice and its disappearance from the cell surface after warming cells to 37 °C followed using flow cytometry. Despite the fact that lysosomes were severely fragmented, transferrin was internalized at the same rate in TBC1D15 over-expressing and control cells (Fig. 3A). Transferrin internalization was delayed, however, in cells expressing dominant-negative Rab5-S34N. For recycling assays, FL5.12 cells were maintained in media containing biotinylated transferrin for 1 h at 37 °C. The cells were then washed, placed in media containing excess unlabeled transferrin, and the clearance of biotinylated transferrin from cells as a result of recycling measured over time. While a GDP-locked mutant of Rab11 reduced the rate of transferrin recycling, cells overexpressing TBC1D15 recycled transferrin with kinetics that were indistinguishable from controls (Fig. 3B). These experiments indicate that in intact cells, TBC1D15 functions as a GAP for Rab7, but not Rab4, 5, or 11. We have therefore renamed TBC1D15 Rab7-GAP.

mVps39 Does Not Display the Properties of a Rab7 GEF

Yeast Vps39 mutants possess a fragmented vacuole (26, 27, 60, 61). This may be because Vps39 functions as a GEF for the yeast ortholog of Rab7, Ypt7 (23). However, the GEF activity of Vps39 remains controversial. To test whether mVps39 can function as a Rab7 GEF, the effect of mVps39 overexpression on Rab7-GTP loading, lysosomal morphology, and growth factor withdrawal-induced apoptosis was evaluated. A mVps39 cDNA was derived from FL5.12 cells by RT-PCR. An N-terminal Myc epitope tag was added to track the protein as other groups have established that this modification does not interfere with Vps39 function (28, 42). Two splicing variants of mVps39 are expressed in cells. The cDNA we isolated from FL5.12 cells corresponded to the shorter isoform of Vps39 that lacks exon 3 resulting in the replacement of 12 amino acids in the longer isoform with a glycine residue. Based on the published sequence of mVps39, four point mutations had been introduced during the process of RT-PCR (Fig. 4A). Rather than repairing this construct or screening additional clones, we obtained the wild-type mVps39 cDNA from Dr. Robert Piper's group (41, 43). Because the mutations in our initial construct were in highly conserved domains of the protein (Fig. 4, A and B), we included the mutant mVps39 (mVps39_mut) in subsequent assays.

If mVps39 functions as a Rab7 GEF, overexpressing mVps39 should increase the fraction of Rab7 that is GTP-bound. However, wild-type mVps39 did not alter Rab7-GTP levels (Fig. 1, C and D). To confirm that this mVps39 construct was fully functional, its ability to alter lysosomal morphology in HeLa cells was evaluated. Consistent with previous reports (42, 43), transient expression of mVps39 in HeLa cells produced lysosomal clustering in 50% of the transfected cells, a 10-fold increase over background (Fig. 4, C and D). Lysosomal clustering is not readily detected in FL5.12 cells as they normally possess very few lysosomes (Fig. 2, A and B). Intriguingly, mVps39_mut appeared to act as a dominant-negative protein. When introduced into HeLa cells, mVps39_mut did not cluster Lamp1-positive structures (Fig. 4, C and D). Similarly, mVps39_mut did not alter Rab7 GTP binding (Fig. 1, C and D). However, mVps39_mut produced lysosomal fragmentation of a similar magnitude to that seen with the dominant-negative Rab7-T22N mutant when expressed in FL5.12 cells (Fig. 2, A and B). Taken together, these data strongly suggest that mVps39 does not promote lysosomal clustering or fusion by functioning as a Rab7 GEF.

In keeping with its effects on lysosomal morphology, mVps39_mut dramatically increased growth factor-independent cell survival (Fig. 4E). Wild-type mVps39 neither fragmented the lysosome nor increased growth factor-independent cell survival (Figs. 2, A and B and 4F). Lysosomal fusion reactions are required for cells to derive bioenergetic benefits from autophagy (7, 62). Cell lines over-expressing TBC1D15 or mVps39_mut were not resistant to nutrient withdrawal (Fig. 4G). In contrast, cells expressing a Bcl-2 mutant, G145A, that does not interfere with autophagy (63) were resistant to nutrient deprivation-induced death. Thus, the growth factor-independent cell survival seen in cell lines expressing Rab7-GAP or mVps39_mut did not result from a primary apoptotic defect.

How mVps39_mut altered lysosomal morphology was not clear. mVps39_mut did not interfere with Rab7 recruitment onto endosomal membranes; all Lysotracker Red-positive structures were GFP-Rab7-positive in control cells and in cells expressing mVps39_mut (Fig. 4H). mVps39 facilitates the exchange of Rab5 for Rab7 on endosomes, mVps39 RNAi induces swollen Rab5-positive endosomes (44). However, GFP-Rab5 localization was indistinguishable in control and mVps39_mut-expressing FL5.12 cells (data not shown). Loss of acidification blocks vacuole fusion in yeast (64). Based on their strong staining with Lysotracker Red, the fragmented lysosomes in mVps39_mut-expressing cells appeared to be acidified. Because mVps39_mut fragmented lysosomes (Fig. 2, A and B) without affecting Rab7 GTP binding status (Fig. 1, C and D) reduced Rab7 GEF activity is also unlikely. If mVps39 functions as a Rab7 effector, mVps39_mut might block the downstream effects of Rab7 activation. In this case, mVps39 would be expected to preferentially bind to the GTP-bound form of Rab7. Using a variety of detergents and buffers, we were unable to detect an association between mVps39 and Rab7 or Rab7-Q67L via co-immunoprecipitation. We were likewise unable to isolate mVps39 from cell lysates using recombinant GST-Rab7 or GST-Rab7-Q67L immobilized on glutathione beads. Taken together, these results suggest that mVps39_mut alters lysosomal morphology through an indirect or even Rab7-independent action.