Purification and functional properties of a Rab8-specific GEF (Rabin3) in action remodeling and polarized transport.
Considering the large number of Rab proteins, only a few Rab-specific exchange factors have been found and characterized. Rab8 is involved in mediating polarized membrane traffic through reorganization of actin and microtubules. It is possible to use the yeast two-hybrid technique to find potential Rab activators. A human protein (Rabin8) and its rat equivalent (Rabin3) were found to bind Rab8 and function as nucleotide exchange factors for Rab8 but not for Rab3A and Rab5. Endogenous and ectopically expressed Rabin8 frequently colocalize with cortical actin. This association is increased by cytochalasin D and phorbol esters that also induced the translocation of both Rabin8 and Rab8 to lamellipodia-like structures. We also show that a GFP-fused Rabin8 behaves identically in this respect. Furthermore, coexpression of Rabin8 with the dominant negative mutant of Rab8 leads to translocation of Rabin8 onto vesicular structures enriched in cell protrusions, indicating that both Rab8 and Rabin8 are involved in mediating polarized membrane transport. This chapter presents a detailed description of the methods and protocols developed to find and characterize a Rab8-specific activator.
Katarina
K
Hattula
Johan
J
Peränen
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D000081410:Germinal Center Kinases; D020662:Guanine Nucleotide Exchange Factors; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D017931:DNA Primers; D000081410:Germinal Center Kinases; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; C092115:Rab3ip protein, rat; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases