Sed4p functions as a positive regulator of Sar1p probably through inhibition of the GTPase activation by Sec23p.
BACKGROUND
Sar1p belongs to a unique subfamily of small GTPases and is essential for formation of the transport vesicles from the endoplasmic reticulum (ER) that are destined to the Golgi apparatus. To understand how the GTPase cycle of Sar1p is regulated, we screened for multicopy suppressors of sar1 ts mutants and identified SED4.


RESULTS
Although deletion of sed4 alone shows no growth defect, sar1 delta(sed4) double mutant cells are inviable. In the delta(sed4) background, the suppression activity of SAR1 towards sec12 and sec16 is lost. These observations suggest that SED4 is a very close partner of SAR1 and imply that Sed4p may act to increase the active Sar1p in the cell. Over-expression of SEC12 does not remedy the lethality of sar1 delta(sed4). The purified cytoplasmic domain of Sed4p does not show a guanine nucleotide exchange (GEF) activity toward Sar1p nor increase the GEF activity of Sec12p. On the contrary, over-expression of SED4 aggravates the ts growth of sec23 cells. The cytoplasmic domain of Sed4p weakly inhibits the GTPase-activating (GAP) activity of Sec23p toward Sar1p. In a microsome-based COPII binding assay, the binding of the GDP-form mutant Sar1p (D32G) is lower on the delta(sed4) microsomes than on the wild-type membranes.


CONCLUSIONS
We propose a model that Sed4p counteracts the GAP action of Sec23p on to Sar1p.
Saito-Nakano
Y
Y
Molecular Membrane Biology Laboratory, RIKEN, Wako, Saitama 351-0198, Japan.
Nakano
A
A
D022181:COP-Coated Vesicles; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008954:Models, Biological; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins
D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; C079037:SEC23 protein, S cerevisiae; C096368:SED4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D020559:Monomeric GTP-Binding Proteins; C077395:SAR1 protein, S cerevisiae