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Myristoylation of ARF family GTPases is required for their association with Golgi and endosomal membranes, where they regulate protein sorting and the lipid composition of these organelles. The Golgi-localized ARF-like GTPase Arl3p/ARP lacks a myristoylation signal, indicating that its targeting mechanism is distinct from myristoylated ARFs. We demonstrate that acetylation of the N-terminal methionine of Arl3p requires the NatC N(alpha)-acetyltransferase and that this modification is required for its Golgi localization. Chemical crosslinking and fluorescence microscopy experiments demonstrate that localization of Arl3p also requires Sys1p, a Golgi-localized integral membrane protein, which may serve as a receptor for acetylated Arl3p.

NatB Nalpha-terminal acetyltransferase of Saccharomyces cerevisiae acts cotranslationally on proteins with Met-Glu- or Met-Asp- termini and subclasses of proteins with Met-Asn- and Met-Met- termini. NatB is composed of the interacting Nat3p and Mdm20p subunits, both of which are required for acetyltransferase activity. The phenotypes of nat3-Delta and mdm20-Delta mutants are identical or nearly the same and include the following: diminished growth at elevated temperatures and on hyperosmotic and nonfermentable media; diminished mating; defective actin cables formation; abnormal mitochondrial and vacuolar inheritance; inhibition of growth by DNA-damaging agents such as methyl methanesulfonate, bleomycin, camptothecin, and hydroxyurea; and inhibition of growth by the antimitotic drugs benomyl and thiabendazole. The similarity of these phenotypes to the phenotypes of certain act1 and tpm1 mutants suggests that such multiple defects are caused by the lack of acetylation of actin and tropomyosins. However, the lack of acetylation of other unidentified proteins conceivably could cause the same phenotypes. Furthermore, unacetylated actin and certain N-terminally altered actins have comparable defective properties in vitro, particularly actin-activated ATPase activity and sliding velocity.

The evolutionarily conserved Mdm20 protein (Mdm20p) plays an important role in tropomyosin-F-actin interactions that generate actin filaments and cables in budding yeast. However, Mdm20p is not a structural component of actin filaments or cables, and its exact function in cable stability has remained a mystery. Here, we show that cells lacking Mdm20p fail to N-terminally acetylate Tpm1p, an abundant form of tropomyosin that binds and stabilizes actin filaments and cables. The F-actin-binding activity of unacetylated Tpm1p is reduced severely relative to the acetylated form. These results are complemented by the recent report that Mdm20p copurifies with one of three acetyltransferases in yeast, the NatB complex. We present genetic evidence that Mdm20p functions cooperatively with Nat3p, the catalytic subunit of the NatB acetyltransferase. These combined results strongly suggest that Mdm20p-dependent, N-terminal acetylation of Tpm1p by the NatB complex is required for Tpm1p association with, and stabilization of, actin filaments and cables.

Formins have been implicated in the regulation of cytoskeletal structure in animals and fungi. Here we show that the formins Bni1 and Bnr1 of budding yeast stimulate the assembly of actin filaments that function as precursors to tropomyosin-stabilized cables that direct polarized cell growth. With loss of formin function, cables disassemble,whereas increased formin activity causes the hyperaccumulation of cable-like filaments. Unlike the assembly of cortical actin patches, cable assembly requires profilin but not the Arp2/3 complex. Thus formins control a distinct pathway for assembling actin filaments that organize the overall polarity of the cell.

The actin cytoskeleton is required for many aspects of cell division in yeast, including mitochondrial partitioning into growing buds (mitochondrial inheritance). Yeast cells lacking MDM20 function display defects in both mitochondrial inheritance and actin organization, specifically, a lack of visible actin cables and enhanced sensitivity to Latrunculin A. mdm20 mutants also exhibit a temperature-sensitive growth phenotype, which we exploited to isolate second-site suppressor mutations. Nine dominant suppressors selected in an mdm20/mdm20 background rescue temperature-sensitive growth defects and mitochondrial inheritance defects and partially restore actin cables in haploid and diploid mdm20 strains. The suppressor mutations define new alleles of ACT1 and TPM1, which encode actin and the major form of tropomyosin in yeast, respectively. The ACT1 mutations cluster in a region of the actin protein predicted to contact tropomyosin, suggesting that they stabilize actin cables by enhancing actin-tropomyosin interactions. The characteristics of the mutant ACT1 and TPM1 alleles and their potential effects on protein structure and binding are discussed.

In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament-binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20 delta cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton.

We report that expression of a nearly full-length cDNA clone of the L-A double-stranded RNA virus causes virus loss in a wild-type strain of Saccharomyces cerevisiae. We show that in this system exclusion of the L-A virus is independent of the presence of the packaging site or of cis sites for replication and transcription and completely dependent on expression of functional recombinant gag and gag-pol fusion protein. Thus, this exclusion is not explained in terms of overexpression of packaging signals. Mutation of the chromosomal SKI2 gene, known to repress the copy number of double-stranded RNA cytoplasmic replicons of S. cerevisiae, nearly eliminates the exclusion. We suggest that exclusion is due to competition by proteins expressed from the plasmid for a possibly limiting cellular factor. Our hypotheses on exclusion of L-A proteins may also apply to resistance to plant viruses produced by expression of viral replicases in transgenic plants.

The MAK10 gene is necessary for the propagation of the L-A dsRNA virus of the yeast Saccharomyces cerevisiae. We have isolated MAK10 from selected phage lambda genomic DNA clones that map near MAK10. This gene encodes a 733-amino acid protein with several regions of similarity to T cell receptor alpha-subunit V (variable) regions. We show that MAK10 is essential for optimal growth on nonfermentable carbon sources independent of its effect on L-A. Although loss of L-A by mak10-1 mutants is partially suppressed by loss of the mitochondrial genome, no such suppression of a mak10::URA3 mutation was observed. Using MAK10-lacZ fusions we show that MAK10 is expressed at a very low level and that it is glucose repressed. The highest levels of expression were seen in tup1 and cyc8 mutants, known to be defective in glucose repression. These results suggest that the mitochondrial genome and L-A dsRNA compete for the MAK10 protein.