The Rab-interacting lysosomal protein (RILP) regulates vacuolar ATPase acting on the V1G1 subunit

Abstract

There was an error published in J. Cell Sci. 127, 2697-2708.In Fig. 2, the V1C1 western blot was inadvertently duplicated in panels A and B. The V1C1 western blot has been replaced with the correct image in panel B in the figure shown below. There are no changes to the figure legend, which is accurate. This error does not affect the conclusions of the study.The authors apologise to the readers for any confusion that this error might have caused. or cells expressing HA-RILP were subjected to western blot analysis using primary antibodies against V1G1, HA, V1C1, V0D1 and tubulin. (B) Cells treated with control siRNA (scr), RILP-specific siRNA (RILPi) or V1G1-specific siRNA (V1G1i) were subjected to western blot analysis using primary antibodies against V1G1, RILP, V1C1, V0D1 and tubulin. (C) Lysates of control HeLa cells or cells expressing HA-RILP, HA-RILPΔC2 or HA-RILPC33 were subjected to western blot analysis using anti-V1G1, anti-HA and anti-tubulin antibodies. (D) Cells treated with control siRNA or RILP-specific siRNA and transfected with HA-RILP, as indicated, were subjected to western blot analysis using anti-V1G1, anti-RILP and anti-tubulin antibodies. (E-G) Real-time PCR was performed on control HeLa cells, or cells expressing HA-RILP (RILP1 and RILP2) or silenced for RILP (RILPi1 and RILPi2), and the amount of RILP (E,F) or of V1G1 (G) was quantified relative to the control GAPDH RNA transcript. (H) Control cells or cells expressing HA-RILP, as indicated, were either untreated or treated with the proteasomal inhibitor MG132. Lysates were then subjected to western blot analysis with antibodies against V1G1, HA and tubulin. (I) Control cells or cells transfected with HA-V1G1 and RILP, as indicated, were either untreated or treated with MG132. Lysates were subjected to western blot analysis using antibodies against RILP, V1G1 and tubulin, and to immunoprecipitation using an anti-HA antibody. Immunoprecipitates were subjected to western blot analysis using an anti-ubiquitin antibody. BSTRACTRab-interacting lysosomal protein (RILP) is a downstream effector of the Rab7 GTPase. GTP-bound Rab7 recruits RILP to endosomal membranes and, together, they control late endocytic traffic, phagosome and autophagosome maturation and are responsible for signaling receptor degradation. We have identified, using different approaches, the V1G1 (officially known as ATP6V1G1) subunit of the vacuolar ATPase (V-ATPase) as a RILP-interacting protein. V1G1 is a component of the peripheral stalk and is fundamental for correct V-ATPase assembly. We show here that RILP regulates the recruitment of V1G1 to late endosomal and lysosomal membranes but also controls V1G1 stability. Indeed, we demonstrate that V1G1 can be ubiquitylated and that RILP is responsible for proteasomal degradation of V1G1. Furthermore, we demonstrate that alterations in V1G1 expression levels impair VATPase activity. Thus, our data demonstrate for the first time that RILP regulates the activity of the V-ATPase through its interaction with V1G1. Given the impor...

A type 2 diabetes disease module with a high collective influence for Cdk2 and PTPLAD1 is localized in endosomes

“…The fact that V-ATPase controls the activity of AMPK [63] emphasizes the idea that all the conditions are present in IREN to auto-regulate this node and thus IR routing, signaling and hepatic clearance in relation to global cell energy status. The presence of the V-ATPase inhibitor RILP [46] in IR immunoprecipitates, which was nearly abolished by PTPLAD1 deletion (Fig 4D), further supports the idea that PTPLAD1 has a large capacity for action to decide IR routing towards early versus late compartments [45].…”

“…We observed an insulin-dependent recruitment of the Rab- interacting lysosomal protein (RILP) in IR immunoprecipitates that was nearly abolished by PTPLAD1 deletion (Fig 4D). RILP was demonstrated to be required for EGFR confinement and degradation in late endosome compartments [45] and is an inhibitor of V-ATPase activity [46]. This supports the idea of a key role for PTPLAD1 in early events of IR internalisation and recycling via RAB5c, Rab11a and actin cytoskeleton elements.…”

Characterization of the role of RILP in cell migration

“…Transfection of cells with siRNA was performed using RNAiMAX from Invitrogen following the manufacturer’s instructions. RILP siRNA efficiency in silencing was reported previously: 22 sense sequence 5’-GAUCAAGGCCAAGAUGUUATT- 3’ and antisense sequence 5’-UAACAUCUUGGCCUUGAUCTT- 3’. As a negative control we used a control RNA: sense sequence 5’-ACUUCGAGCGUGCAUGGCUTT- 3’ and antisense sequence 5’-AGCCAUGCACGCUCGAAGUTT-3’.…”

“…Rab7a and RILP could regulate migration independently using different mechanisms and the migration outcome could be the result of the opposite contribution of the two proteins. Alternatively, as recently demonstrated for a newly discovered regulation mechanism of V-ATPase mediated by RILP, 22,37 the interaction between Rab7a and RILP could modulate the availability of RILP (or of Rab7a) to regulate migration.…”

Folliculin – A tumor suppressor at the intersection of metabolic signaling and membrane traffic

“…This in turn impacts on the degradative capacity of the Cathepsin L protease. RILP was recently reported to interact directly with the vacuolar ATPase and regulate its activity, suggesting a potential mechanism that may couple positioning and acidity43 .The distribution of lysosomes also affects their interaction with other organelles.A recent report suggested Rab34 may respond to extrinsic stimuli (lipopolysaccharide) to promote context-dependent changes in lysosome distribution to modulate lysosome fusion with phagosomes and control antigen cross presentation in dendritic cells44 ; it would be interesting to determine whether FLCN may also contribute to this pathway. Similarly, elevation of intracellular calcium triggers lysosome exocytosis to participate in plasma membrane repair 45 and blocking lysosome transport to the cell periphery reduces cell spreading, migration and cancer cell invasion 30,46 .…”