Dataset Information

Rab coupling protein is selectively degraded by calpain in a Ca2+-dependent manner.

ABSTRACT: RCP (Rab coupling protein) belongs to the recently identified Rab11-FIPs (Rab11 family of interacting proteins). All the Rab-FIP members have the ability to bind Rab11 tightly via a Rab-binding domain located near their C-termini. RCP belongs to the class I Rab11-FIP subfamily, characterized by the presence of a conserved C2 domain near its N-terminus. The function of this protein in Rab11-dependent membrane trafficking remains to be fully understood. In the present study, we have identified three putative PEST (Pro, Glu, Ser/Thr-rich) sequences in RCP. PEST motifs play a role in targeting a protein for proteolytic degradation. We have demonstrated that RCP undergoes calcium-dependent degradation which can be prevented by specific calpain inhibitors. Using a mutant, lacking the three PEST sequences, RCP(DeltaPEST), we demonstrated that they are necessary for the cleavage of RCP by calpains. When expressed in A431 cells, RCP(DeltaPEST) displays significantly greater localization to the plasma membrane, compared with the wild-type protein. Similarly, treatment with the calpain inhibitor, calpeptin, results in the redistribution of endogenous RCP to the periphery of the cell. We propose that once the Rab11/RCP-regulated cargo has been delivered from the endocytic recycling compartment to the plasma membrane, RCP is inactivated by calpain-mediated proteolysis.

SUBMITTER: Marie N

PROVIDER: S-EPMC1184555 | biostudies-literature | 2005 Jul

REPOSITORIES: biostudies-literature

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Rab coupling protein is selectively degraded by calpain in a Ca2+-dependent manner.

Marie Nicolas N Lindsay Andrew J AJ McCaffrey Mary W MW

The Biochemical journal 20050701 Pt 1

RCP (Rab coupling protein) belongs to the recently identified Rab11-FIPs (Rab11 family of interacting proteins). All the Rab-FIP members have the ability to bind Rab11 tightly via a Rab-binding domain located near their C-termini. RCP belongs to the class I Rab11-FIP subfamily, characterized by the presence of a conserved C2 domain near its N-terminus. The function of this protein in Rab11-dependent membrane trafficking remains to be fully understood. In the present study, we have identified thr ...[more]

PMID: 15769254

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Project description:Mutations in the PKD1 gene result in autosomal dominant polycystic kidney disease (ADPKD), the most common monogenetic cause of end-stage renal disease (ESRD) in humans. Previous reports suggested that PKD1, together with PKD2/polycystin-2, may function as a receptor-cation channel complex at cilia and on intracellular membranes and participate in various signaling pathways to regulate cell survival, proliferation and macroautophagy/autophagy. However, the exact molecular function of PKD1 and PKD2 has remained enigmatic. Here we used Pkd1-deficient mouse inner medullary collecting duct cells (mIMCD3) genetically deleted for Pkd1, and tubular epithelial cells isolated from nephrons of doxycycline-inducible conditional pkd1fl/fl;Pax8rtTA;TetOCre+ knockout mice to show that the lack of Pkd1 caused diminished lysosomal acidification, LAMP degradation and reduced CTSB/cathepsin B processing and activity. This led to an impairment of autophagosomal-lysosomal fusion, a lower delivery of ubiquitinated cargo from multivesicular bodies (MVB)/exosomes to lysosomes and an enhanced secretion of unprocessed CTSB into the extracellular space. The TFEB-dependent lysosomal biogenesis pathway was however unaffected. Pkd1-deficient cells exhibited increased activity of the calcium-dependent CAPN (calpain) proteases, probably due to a higher calcium influx. Consistent with this notion CAPN inhibitors restored lysosomal function, CTSB processing/activity and autophagosomal-lysosomal fusion, and blocked CTSB secretion and LAMP degradation in pkd1 knockout cells. Our data reveal for the first time a lysosomal function of PKD1 which keeps CAPN activity in check and ensures lysosomal integrity and a correct autophagic flux.Abbreviations: acCal: acetyl-calpastatin peptide; ADPKD: autosomal dominant polycystic kidney disease; CI-1: calpain inhibitor-1; CQ: chloroquine; Dox: doxycycline; EV: extracellular vesicles; EXO: exosomes; LAMP1/2: lysosomal-associated membrane protein 1/2; LGALS1/GAL1/galectin-1: lectin, galactose binding, soluble 1; LMP: lysosomal membrane permeabilization; mIMCD3: mouse inner medullary collecting duct cells; MV: microvesicles; MVB: multivesicular bodies; PAX8: paired box 8; PKD1/polycystin-1: polycystin 1, transient receptor potential channel interacting; PKD2/polycystin-2: polycystin 2, transient receptor potential cation channel; Tet: tetracycline; TFEB: transcription factor EB; VFM: vesicle-free medium; WT: wild-type.

Dataset Information

Rab coupling protein is selectively degraded by calpain in a Ca2+-dependent manner.

Publications

Rab coupling protein is selectively degraded by calpain in a Ca2+-dependent manner.

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