Molecular characterization of Rab11 interactions with members of the family of Rab11-interacting proteins.
The Rab11 subfamily of GTPases plays an important role in vesicle trafficking from endosomes to the plasma membrane. At least six Rab11 effectors (family of Rab11-interacting proteins (FIPs)) have been shown to interact with Rab11 and are hypothesized to regulate various membrane trafficking pathways such as transferrin recycling, cytokinesis, and epidermal growth factor trafficking. In this study, we characterized interactions of FIPs with the Rab11 GTPase using isothermal titration calorimetric studies and mutational analysis. Our data suggest that FIPs cannot differentiate between GTP-bound Rab11a and Rab11b in vitro (50-100 nm affinity) and in vivo. We also show that, although FIPs interact with the GDP-bound form of Rab11 in vitro, the binding affinity (>1000 nm) is not sufficient for FIP and GDP-bound Rab11 interactions to occur in vivo. Mutational analysis revealed that both the conserved hydrophobic patch and Tyr628 are important for the GTP-dependent binding of Rab11 to FIPs. The entropy and enthalpy analyses suggest that binding to Rab11a/b may induce conformational changes in FIPs.
Junutula
Jagath R
JR
Genentech, Incorporated, South San Francisco, California 94080, USA.
Schonteich
Eric
E
Wilson
Gayle M
GM
Peden
Andrew A
AA
Scheller
Richard H
RH
Prekeris
Rytis
R
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D017124:Conserved Sequence; D019281:Dimerization; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014162:Transfection; D014168:Transferrin / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
D002352:Carrier Proteins; D049452:Green Fluorescent Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D006165:Guanylyl Imidodiphosphate; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C463963:RAB11FIP2 protein, human