Rab coupling protein is selectively degraded by calpain in a Ca2+-dependent manner.
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.
Marie
Nicolas
N
Department of Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland.
Lindsay
Andrew J
AJ
McCaffrey
Mary W
MW
D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002154:Calpain / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000502:physiology; D017124:Conserved Sequence; D004151:Dipeptides / Q000494:pharmacology; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D022142:Secretory Vesicles / Q000502:physiology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004151:Dipeptides; D008565:Membrane Proteins; C488966:RAB11FIP1 protein, human; C071482:calpeptin; D002154:Calpain; D002118:Calcium