The small GTPase Rab32 resides on lysosomes to regulate mTORC1 signaling
Authors: Kristina Drizyte-Miller
Micah B Schott
Hong Cao
Mark A McNiven
Jing Chen
Journal: Journal of cell science (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Lysosome; S6K; Small Rab GTPase; TFEB; mTORC1.
Affiliations: Biochemistry and Molecular Biology Program, Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, 200 1st Street SW, Rochester, MN 55905, USA, Center for Basic Research in Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic, 200 1st Street SW, Rochester, MN 55905, USA, Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 1st Street SW, Rochester, MN 55905, USA

Abstract:  Epithelial cells, such as liver-resident hepatocytes, rely heavily on the Rab family of small GTPases to perform membrane trafficking events that dictate cell physiology and metabolism. Not surprisingly, disruption of several Rab proteins can manifest in metabolic diseases or cancer. Rab32 is expressed in many secretory epithelial cells but its role in cellular metabolism is virtually unknown. In this study, we find that Rab32 associates with lysosomes and regulates proliferation and cell size of Hep3B hepatoma and HeLa cells. Specifically, we identify that Rab32 supports the mechanistic target of rapamycin complex 1 (mTORC1) signaling under basal and amino acid-stimulated conditions. Consistent with inhibited mTORC1, an increase in nuclear TFEB localization and lysosome biogenesis is also observed in Rab32-depleted cells. Finally, we find that Rab32 interacts with mTOR kinase, and that loss of Rab32 reduces the association of mTOR and mTORC1 pathway proteins with lysosomes, suggesting that Rab32 regulates lysosomal mTOR trafficking. In summary, these findings suggest that Rab32 functions as a novel regulator of cellular metabolism through supporting mTORC1 signaling.This article has an associated First Person interview with the first author of the paper. 

Retracted: No

PubPeer comments:
 The authors claim here that * loss of Rab32 reduces the association of mTOR and mTORC1 pathway proteins with lysosomes, suggesting that Rab32 regulates lysosomal mTOR trafficking *, however the data supporting this claim are rather weak (if not missing). Colocalization analyses of mTOR with lysosomal markers (e.g. LAMP1 or LAMP2), using confocal microscopy are the gold-standard to test mTOR shuttling between lysosomes and the cytosol. Such experiments are missing from this paper. Most importantly, the biochemical lysosome-isolation experiments (Sabatini s Lyso-IP system) that the authors use to claim changes with in the lysosomal association of mTOR seem to be rather inconclusive: In Fig.7A, the author claim that less mTOR and Raptor is associated with lysosomes when Rab32 is knocked down. This conclusion cannot be made because the siRab32 cells have less TMEM192-HA containing lysosomes, therefore the authors simply IP less bait . It would be expected then to also co-purify less mTOR and Raptor. This would also be supported by the fact that these samples contain less RagC and Lamtor1 proteins, whose abundance in the lysosomal fraction should normally not be affected (Lamtor1 is tethered to the lysosomal membrane via a lipid modification). Similarly, in 7B the authors normalize all proteins to Lamp1 and not TMEM192 signal. Given that Rab32 knockdown cells show increased lysosomal biogenesis, it is expected that Lamp1 would be upregulated and therefore is not a good normalization control. How would these quantifications look if the proteins would be normalized to TMEM192-HA instead? ![file] In the absence of a conclusive biochemical experiment and a proper microscopy-based method to look at mTOR lysosomal localization, the conclusion that Rab32 regulates mTOR localization is not supported by the data. , 
 Dear Anonymous, Thank you for your interest in our paper. Overall, we find that Rab32 localizes with mTOR on lysosomes, and that knockdown of Rab32 inhibits both basal and AA-stimulated mTOR activity. Consistent with this, Rab32 knockdown caused a dramatic decrease in cell size and proliferation, as well as an increase in TFEB nuclear localization and lysosome biogenesis. Mechanistically, there is much we still donu2019t understand. Our working model is that Rab32 knockdown inhibits mTOR recruitment to the lysosome, and may also alter the lysosomal proteome more generally, as we noted in our discussion. Evidence for reduced mTOR recruitment comes not only from biochemical analysis (i.e. Lyso-IP), but also from the finding that expression of active Rag heterodimers rescues the mTOR signaling defect in Rab32-depleted cells (Fig. 7C,D). The Lyso-IP experiments were done using transiently-transfected cells which required considerable optimization to achieve similar levels of TMEM192-HA, as well as Lamp1, in the pulldown. Normalizing our quantitation to TMEM192-HA did not change the conclusion, as mTOR, Raptor, and Lamtor1 are significantly decreased in the Lyso-IP from Rab32-depleted cells, similar to Fig. 7B. I agree that confocal analyses would have strengthened our working model, but unfortunately we ran into several issues as Kristina pursued this with great rigor. First, the dramatic reduction in cell size made any attempts at colocalization analyses (Pearsonu2019s, Manderu2019s) unreliable. Any two markers we chose, even nonspecific markers of various organelles, showed increased colocalization presumably due to smaller cell size. We tried Raptor-GFP as well, but this was completely cytosolic in multiple cell lines, plus or minus amino acids. A third problem was that, inexplicable to us, the immunofluorescence labeling is often brighter in Rab32-depleted cells. This occurs irrespective of the antigen or antibody, and does not match Western blot analyses of cell lysates from the very same dish. This is very interesting to us, but is currently beyond our understanding as to why this occurs. Due to these issues, we were not comfortable including our confocal analyses in this study. Best wishes, Micah , 
 #2 Dear Author, since Lamp1 levels are apparently very similar between lanes 2 and 3 of the Lyso-IP samples, and TMEM-HA levels are approximately half in lane 3 compared to lane 2, I find it still very surprising that the quantification of mTOR, Raptor, RagC, Lamtor1 to TMEM would look similar to the quantification of the same proteins to Lamp1. If the authors would like to provide the raw numbers of the quantification to each of the two proteins, this could clarify this issue. By the way, which method was used to quantify these bands? As mentioned in comment #1, and as shown in Fig. 4F,G of this paper, siRab32 increases Lamp1 levels by more than two fold. ![file] By definition, a normalization control is a protein that is not changing between the conditions tested. Therefore, Lamp1 is not a good control for the Lyso-IP experiment. Moreover, in any IP experiment, the levels of a co-IPing protein are quantified per molecule (or per unit) of the bait protein (in this case the TMEM192-HA protein). If both the bait protein and the co-IPing proteins change to the same extent between two conditions, then the binding affinity stays the same. This would also explain why all proteins tested show lower levels in lane 3 compared to lane 2 of the Lyso-IP (in other words, if there is less TMEM pulled-down, this would also lead to less of the co-IPing proteins being pulled-down). Did the authors see any other proteins (not involved in lysosomal biogenesis) that don t change between those two conditions? Regarding the active Rag overexpression experiment (Fig. 7C,D), that the author mentions in their reply as an argument supporting changes in the lysosomal localization of mTOR in siRab32 samples, the actual data would suggest that the Rag overexpression does not really rescue the Rab32 knock-down effect, as it causes a minuscule increase in p-S6K over the baseline levels (compare lane 8 to lane 4 in Fig. 7C). ![file] Unfortunately, lanes 3 and 4 are omitted from the quantification (Fig. 7D), therefore a direct quantitative comparison to assess the effect of Rag GTPases on the siRab32-mediated downregulation of mTOR activity is not possible. ![file] For all these reasons (and as a colocalization analysis, which is nowadays very strait-forward, is missing), in my humble opinion, the claim that Rab32 regulates mTOR localization to the lysosomes is not supported by these data. ,