Identification and verification of Sro7p as an effector of the Sec4p Rab GTPase.
Effectors are operationally defined as proteins that recognize a specific GTPase preferentially in its GTP-bound conformation. Here we present the use of affinity chromatography to identify potential effectors of Sec4p, the Rab GTPase that controls the final stage of the yeast secretory pathway. We describe the preparation of the Rab protein affinity matrix and the yeast lysate used in the purification. We also describe the methods used to identify and verify one candidate, Sro7p, as a bona fide Sec4p effector. This includes tests of the specificity and efficiency of binding both in vitro and in vivo.
Grosshans
Bianka L
BL
Novartis Institutes for Biomedical Research, Basel, Switzerland.
Novick
Peter
P
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D005982:Glutathione Transferase / Q000235:genetics; D047468:Immunoprecipitation / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; C116430:SRO7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D005982:Glutathione Transferase; C064320:SEC4 protein, S cerevisiae; D020691:rab GTP-Binding Proteins