Diacylglycerol-activated Hmunc13 serves as an effector of the GTPase Rab34.
Hmunc13 is a cytosolic diacylglycerol (DAG)-binding protein, which is upregulated in renal cortical tubule and mesangial cells by hyperglycemia. In response to DAG activation, hmunc13 translocates to the Golgi. To investigate how this may relate to its function, we used a bacterial two-hybrid screen to look for hmunc13-interacting proteins. Full-length Rab34 was specifically isolated from a human kidney cDNA library. Co-expression of the two proteins confirmed Rab34 as a Golgi-associated protein, which was immunoprecipitated from cell lysates by hmunc13. Glutathione S-transferase fusion proteins of WT, Q111L (GTP bound), and T66N (GDP bound) mutants were created, and their GTP-binding activity verified by radioactive overlay assay. Binding of hmunc13 was observed with Q111L, barely detectable with T66N and enhanced with Rab34WT loaded with GTPgammaS compared with GDP loaded. Deletion of munc homolgy domain (MHD)-2, eliminated the hmunc13/Rab34 interaction. The Q111L mutant localized to the Golgi apparatus, but T66N was cytosolic. Localization of both mutants and Rab34WT was unchanged by DAG activation. The data suggest that DAG activation of hmunc13 causes it to be translocated to the Golgi, where it binds to GTP-bound Rab34 via MHD-2. Because Rab34 is known to regulate intracellular lysosome positioning, we propose that hmunc13 serves as an effector of Rab34, mediating lysosome-Golgi trafficking.
Speight
Pam
P
Department of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Silverman
Mel
M
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004075:Diglycerides / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D004075:Diglycerides; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C485548:UNC13B protein, human; D015240:Phorbol 12,13-Dibutyrate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C474116:RAB39A protein, human; D020691:rab GTP-Binding Proteins