Depolarization of the actin cytoskeleton is a specific phenotype in Saccharomyces cerevisiae.

The yeast actin cytoskeleton is polarized during most of the cell cycle. Certain environmental factors and mutations are associated with depolarization of the actin cytoskeleton. Is depolarization of the actin cytoskeleton a specific response, or is it a nonspecific reaction to harsh conditions or poor metabolism? If depolarization is a nonspecific response, then any mutation that slows growth should induce depolarization. In addition, the number of genes with the depolarization phenotype should constitute a relatively large part of the genome. To address this question, we determined the effect of slow growth on the actin cytoskeleton, and we determined the frequency of mutations that affect the actin cytoskeleton. Eight mutants with slow growth showed no defect in actin polarization, indicating that slow growth alone is not sufficient to cause depolarization. Among 273 viable haploids disrupted for ORFs of chromosome I and VIII and 950 viable haploids with random genome disruptions, none had depolarization of the cytoskeleton. We conclude that depolarization of the actin cytoskeleton is a specific phenotype.

Localized mutagenesis and evidence for post-transcriptional regulation of MAK3. A putative N-acetyltransferase required for double-stranded RNA virus propagation in Saccharomyces cerevisiae.

The MAK3 gene of Saccharomyces cerevisiae is necessary for the propagation of the L-A double-stranded RNA virus and its satellites, such as M1 that encodes a killer toxin. We cloned the MAK3 gene based on its genetic map position using physically mapped lambda-clones covering nearly all of the yeast genome. The minimal sequence necessary to complement the mak3-1 mutation contained 3 open reading frames (ORFs). Only one (ORF3) was necessary to complement mak3-1. A deletion insertion mutant of ORF3 grew slowly on nonfermentable carbon sources, an effect not due simply to its loss of L-A. Although ORF3 alone is sufficient for MAK3 activity when expressed from an expression vector, in its native context an additional 669 base pairs 3' to the ORF and complementary to the gene for a non-histone protein are necessary for expression, but not for normal steady state transcript levels. This suggests a post-transcriptional control of MAK3 expression by the 3' region. The MAK3 protein has substantial homology with several N-acetyltransferases with consensus patterns h..h.h. . . Y..[HK]GI[AG][KR].Lh. . .h and h.h[DE]. . . .N..A. . .Y . . .GF. . . .. . . .Y . . [DE]G, (h = hydrophobic). Mutation of any of the underlined conserved residues (94GI----AA, 123N----A, 130Y----A, 134GF----SL, 144Y----A, and 149G----A) inactivated the gene, supporting the hypothesis that MAK3 encodes an N-acetyltransferase.