A Rab8-specific GDP/GTP exchange factor is involved in actin remodeling and polarized membrane transport.
The mechanisms mediating polarized delivery of vesicles to cell surface domains are poorly understood in animal cells. We have previously shown that expression of Rab8 promotes the formation of new cell surface domains through reorganization of actin and microtubules. To unravel the function of Rab8, we used the yeast two-hybrid system to search for potential Rab8-specific activators. We identified a coil-coiled protein (Rabin8), homologous to the rat Rabin3 that stimulated nucleotide exchange on Rab8 but not on Rab3A and Rab5. Furthermore, we show that rat Rabin3 has exchange activity on Rab8 but not on Rab3A, supporting the view that rat Rabin3 is the rat equivalent of human Rabin8. Rabin8 localized to the cortical actin and expression of Rabin8 resulted in remodeling of actin and the formation of polarized cell surface domains. Activation of PKC by phorbol esters enhanced translocation of both Rabin8 and Rab8-specific vesicles to the outer edge of lamellipodial structures. Moreover, coexpression of Rabin8 with dominant negative Rab8 (T22N) redistributes Rabin8 from cortical actin to Rab8-specific vesicles and promotes their polarized transport to cell protrusions. The C-terminal region of Rabin8 plays an essential role in this transport. We propose that Rabin8 is a Rab8-specific activator that is connected to processes that mediate polarized membrane traffic to dynamic cell surface structures.
Hattula
Katarina
K
Institute of Biotechnology, Program in Cellular Biotechnology, FIN-00014 University of Helsinki, Finland.
Furuhjelm
Johanna
J
Arffman
Airi
A
Peränen
Johan
J
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001692:Biological Transport; D015152:Blotting, Northern; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D000081410:Germinal Center Kinases; D005982:Glutathione Transferase / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics* / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014018:Tissue Distribution; D014162:Transfection; D020798:Two-Hybrid System Techniques; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D020826:rab3A GTP-Binding Protein / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
D000199:Actins; D000081410:Germinal Center Kinases; D020662:Guanine Nucleotide Exchange Factors; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; C082377:RAB8A protein, human; C513177:Rab8b protein, rat; D020691:rab GTP-Binding Proteins; D020826:rab3A GTP-Binding Protein; D020696:rab5 GTP-Binding Proteins