Mutational Analysis of Saccharomyces cerevisiae ARF1

doi:10.1074/jbc.270.1.143

Mutational Analysis of Saccharomyces cerevisiae ARF1(*)

From the Laboratory of Biological Chemistry, Developmental Therapeutics Program, Division of Cancer Treatment, NCI, National Institutes of Health, Bethesda, Maryland 20892 and the
Department of Biological Sciences, Stanford University, Stanford, California 94305-8309

^§ To whom correspondence should be addressed:
Bldg. 37, Rm. 5D-02, Bethesda, MD 20892
.

Abstract

Wild type and eight point mutants of Saccharomyces cerevisiae ARF1 were expressed in yeast and bacteria to determine the roles of specific residues in in vivo and in vitro activities. Mutations at either Gly² or Asp resulted in recessive loss of function. It was concluded that N-myristoylation is required for Arf action in cells but not for either nucleotide exchange or cofactor activities in vitro. Asp (homologous to Gly of p21) was essential for the binding of the activating nucleotide, guanosine 5′-3-O-(thio)triphosphate. This is in marked contrast to results obtained after mutagenesis of the homologous residue in p21 or G_sα, and suggests a fundamental difference in the guanine nucleotide binding site of Arf with respect to these other GTP-binding proteins.

Two dominant alleles were also identified, one activating dominant ([Q71L]Arf1) and the other ([N126I]) a negative dominant. A conditional allele, [W66R]Arf1, was characterized and shown to have ≈300-fold lower specific activity in an in vitro Arf assay. Two high-copy suppressors of this conditional phenotype were cloned and sequenced. One of these suppressors, SFS4, was found to be identical to PBS2/HOG4, recently shown to encode a microtubule-associated protein kinase kinase in yeast.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

Arf

ADP-ribosylation factor

Arl

Arf-like

GAP

GTPase activating protein

S.D.

synthetic medium with 2% dextrose as carbon source

SGal

synthetic medium with 2% galactose as carbon source

bp

base pair(s)

GTPS

guanosine 5′-3-O-(thio)triphosphate

SFS

suppressor of fluoride sensitivity.
↵² A. Boman and K. Wilson, unpublished observation.
↵³ T. Stearns, M. A. Hoyt, and D. Botstein, unpublished observation.
↵⁴ Two regions of non-identity were discovered when SFS4 was aligned with the published sequence of PBS2. We obtained the original PBS2 plasmid (YEp24.PBS2) from Dr. George Boguslawski, who first described this gene. Sequencing of the regions in dispute revealed errors in the published sequence, resulting in changes in residues 222-223 to Gly-Leu (instead of Ala-Val) and a stop codon after Leu, resulting in a predicted protein that is 42-amino acids shorter (668 residues). The sequence of the 4500-bp SstI-BamHI genomic fragment has been submitted to Genbank and has been assigned the accession number U12237[GenBank].
↵⁵ R. A. Kahn, unpublished observation.
↵⁶ P. A. Randazzo and R. A. Kahn, unpublished observation.
↵⁷ Amor, J. C., Harrison, D. H., Kahn, R. A., and Ringe, D.(1994) Nature, in press.
- Received July 21, 1994.
- Revision received October 31, 1994.