Systematic structure-function analysis of the small GTPase Arf1 in yeast.
Members of the ADP-ribosylation factor (Arf) family of small GTPases are implicated in vesicle traffic in the secretory pathway, although their precise function remains unclear. We generated a series of 23 clustered charge-to-alanine mutations in the Arf1 protein of Saccharomyces cerevisiae to determine the portions of this protein important for its function in cells. These mutants display a number of phenotypes, including conditional lethality at high or low temperature, defects in glycosylation of invertase, dominant lethality, fluoride sensitivity, and synthetic lethality with the arf2 null mutation. All mutations were mapped onto the available crystal structures for Arf1p: Arf1p bound to GDP, to GTP, and complexed with the regulatory proteins ArfGEF and ArfGAP. From this systematic structure-function analysis we demonstrate that all essential mutations studied map to one hemisphere of the protein and provide strong evidence in support of the proposed ArfGEF contact site on Arf1p but minimal evidence in support of the proposed ArfGAP-binding site. In addition, we describe the isolation of a spatially distant intragenic suppressor of a dominant lethal mutation in the guanine nucleotide-binding region of Arf1p.
Click
Eleanor S
ES
Department of Genetics, Stanford University, California 94305, USA.
Stearns
Tim
T
Botstein
David
D
D020823:ADP-Ribosylation Factor 1 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D020727:ADP-Ribosylation Factors / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D020690:GTPase-Activating Proteins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013696:Temperature; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
D020690:GTPase-Activating Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors