Binding and hydrolysis of guanine nucleotides by Sec4p, a yeast protein involved in the regulation of vesicular traffic.
The 23.5-kDa Sec4 protein is required for vesicular transport between the Golgi apparatus and the plasma membrane in Saccharomyces cerevisiae. In order to analyze its biochemical properties, we have purified the soluble pool of the wild-type protein from an overproducing yeast strain. At 30 degrees C, Sec4p bound [35S] guanosine 5'-O-(thiotriphosphate) (GTP gamma S) with a rate of 0.18 min-1 in a reaction requiring micromolar concentration of free magnesium ions. The protein had high affinity for guanine nucleotides with Kd values for GTP gamma S and GTP of 3.7 nM and 3.5 nM, respectively, and that for GDP of 77 nM. The dissociation of [3H] GDP from Sec4p occurred with a rate of 0.21 min-1 suggesting that the association of GTP gamma S was the result of exchange for prebound GDP. The release of GTP from Sec4p was slow and correlated with a low inherent GTPase activity of 0.0012 min-1. By analogy with other classes of GTP binding proteins, both the nucleotide exchange and hydrolysis activities of Sec4p may be modulated in vivo to facilitate its role in the regulation of intercompartmental membrane traffic.
Kabcenell
A K
AK
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
Goud
B
B
Northup
J K
JK
Novick
P J
PJ
D001667:Binding, Competitive; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002793:Cholic Acids; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D005656:Fungal Proteins / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006056:Golgi Apparatus / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D013873:Thionucleotides / Q000378:metabolism; D020691:rab GTP-Binding Proteins
D002793:Cholic Acids; D005656:Fungal Proteins; D006150:Guanine Nucleotides; D029701:Saccharomyces cerevisiae Proteins; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D004492:Edetic Acid; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C064320:SEC4 protein, S cerevisiae; D020691:rab GTP-Binding Proteins; D008274:Magnesium; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate