Novel Interactions between Actin and the Proteasome Revealed by Complex Haploinsufficiency

Saccharomyces cerevisiae has been a powerful model for uncovering the landscape of binary gene interactions through whole-genome screening. Complex heterozygous interactions are potentially important to human genetic disease as loss-of-function alleles are common in human genomes. We have been using complex haploinsufficiency (CHI) screening with the actin gene to identify genes related to actin function and as a model to determine the prevalence of CHI interactions in eukaryotic genomes. Previous CHI screening between actin and null alleles for non-essential genes uncovered ∼240 deleterious CHI interactions. In this report, we have extended CHI screening to null alleles for essential genes by mating a query strain to sporulations of heterozygous knock-out strains. Using an act1Δ query, knock-outs of 60 essential genes were found to be CHI with actin. Enriched in this collection were functional categories found in the previous screen against non-essential genes, including genes involved in cytoskeleton function and chaperone complexes that fold actin and tubulin. Novel to this screen was the identification of genes for components of the TFIID transcription complex and for the proteasome. We investigated a potential role for the proteasome in regulating the actin cytoskeleton and found that the proteasome physically associates with actin filaments in vitro and that some conditional mutations in proteasome genes have gross defects in actin organization. Whole-genome screening with actin as a query has confirmed that CHI interactions are important phenotypic drivers. Furthermore, CHI screening is another genetic tool to uncover novel functional connections. Here we report a previously unappreciated role for the proteasome in affecting actin organization and function

Analysis of CDC3 and CDC12, two genes involved in budding in the yeast Saccharomyces cerevisiae.

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud. Electron microscopic observations suggested that these filaments assemble at the budding site just after bud emergence and disappeared shortly before cytokinesis (B. Byers and L. Goetsch, J. Cell Biol. 69:717-721, 1976). Mutants defective in any of four genes (CDC3, CDC10, CDC11, and CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and cell-wall deposition and an inability to complete cytokinesis. DNA sequence analysis has revealed that these genes encode a family of four similar proteins with predicted molecular weights ranging from 37 to 60 kilodaltons. These proteins show no extensive similarities to any known proteins (including various filament-forming proteins) in the NBRF protein database and do not appear to contain the heptad repeats characteristic of many filament-forming proteins. Analyses of primary structures and of predicted secondary structures do suggest that these proteins contain nucleotide or phosphoryl binding sites. The cloned CDC3 and CDC12 genes were fused to the E. coli lacZ and trpE genes and the resulting fusion proteins were used to raise polyclonal antibodies specific for the CDC3 and CDC12 gene products. In immunofluorescence experiments using affinity-purified antibodies, the neck regions of wild-type and mutant cells stained in patterns consistent with the hypothesis that the CDC3 and CDC12 gene products are constituents of the ring of 10-nm filaments. These antibodies were also used to investigate the timing of localization of these proteins at the budding site. It was found that the CDC3 protein is present through most of the cell cycle while the CDC12 product is usually present only during budding. In addition, it was found that these proteins reorganize in shmooing and mating cells and are present during formation of the first zygotic bud.Ph.D.Biological SciencesMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/128152/2/8812901.pd

Old Yellow Enzyme Protects the Actin Cytoskeleton from Oxidative Stress

Old Yellow Enzyme (OYE) has long served as a paradigm for the study of flavin-containing NADPH oxido-reductases and yet its physiological role has remained a mystery. A two-hybrid interaction between Oye2p and actin led us to investigate a possible function in the actin cytoskeleton. We found that oye deletion strains have an overly elaborate actin cytoskeleton that cannot be attributed to changes in actin concentration but likely reflect stabilization of actin filaments, resulting in excessive actin assembly. Cells expressing the actin mutant act1-123p, which has a weakened interaction with Oye2p, show comparable defects in actin organization to the oye deletion strain that can be suppressed by overexpression of Oye2p. Similarly, mutation of either conserved cysteine of the potential disulfide pair Cys285-Cys374 in actin completely suppresses the actin organization defect of the oyeΔ phenotype. Strains lacking Oye function are also sensitive to oxidative stress as induced by H(2)O(2), menadione, and diamide treatment. Mutation of either Cys285 or Cys374 of actin suppresses the sensitivity of oyeΔ strains to oxidative stress and in fact confers super-resistance to oxidative stress in otherwise wild-type strains. These results suggest that oxidative damage to actin, like that which has been observed in irreversibly sickled red blood cells, may be a general phenomenon and that OYE functions to control the redox state of actin thereby maintaining the proper plasticity of the actin cytoskeleton. In addition to uncovering a long sought biological function for Old Yellow Enzyme, these results establish that cellular sensitivity to oxidative stress can in part be directly attributed to a specific form (C285-C374 disulfide bond formation) of oxidative damage to actin

Modeling complex genetic interactions in a simple eukaryotic genome: actin displays a rich spectrum of complex haploinsufficiencies

Multigenic influences are major contributors to human genetic disorders. Since humans are highly polymorphic, there are a high number of possible detrimental, multiallelic gene pairs. The actin cytoskeleton of yeast was used to determine the potential for deleterious bigenic interactions; ∼4800 complex hemizygote strains were constructed between an actin-null allele and the nonessential gene deletion collection. We found 208 genes that have deleterious complex haploinsufficient (CHI) interactions with actin. This set is enriched for genes with gene ontology terms shared with actin, including several actin-binding protein genes, and nearly half of the CHI genes have defects in actin organization when deleted. Interactions were frequently seen with genes for multiple components of a complex or with genes involved in the same function. For example, many of the genes for the large ribosomal subunit (RPLs) were CHI with act1Δ and had actin organization defects when deleted. This was generally true of only one RPL paralog of apparently duplicate genes, suggesting functional specialization between ribosomal genes. In many cases, CHI interactions could be attributed to localized defects on the actin protein. Spatial congruence in these data suggest that the loss of binding to specific actin-binding proteins causes subsets of CHI interactions

Stable Preanaphase Spindle Positioning Requires Bud6p and an Apparent Interaction between the Spindle Pole Bodies and the Neck▿ †

Faithful partitioning of genetic material during cell division requires accurate spatial and temporal positioning of nuclei within dividing cells. In Saccharomyces cerevisiae, nuclear positioning is regulated by an elegant interplay between components of the actin and microtubule cytoskeletons. Regulators of this process include Bud6p (also referred to as the actin-interacting protein Aip3p) and Kar9p, which function to promote contacts between cytoplasmic microtubule ends and actin-delimited cortical attachment points. Here, we present the previously undetected association of Bud6p with the cytoplasmic face of yeast spindle pole bodies, the functional equivalent of metazoan centrosomes. Cells lacking Bud6p show exaggerated movements of the nucleus between mother and daughter cells and display reduced amounts of time a given spindle pole body spends in close association with the neck region of budding cells. Furthermore, overexpression of BUD6 greatly enhances interactions between the spindle pole body and mother-bud neck in a spindle alignment-defective dynactin mutant. These results suggest that association of either spindle pole body with neck components, rather than simply entry of a spindle pole body into the daughter cell, provides a positive signal for the progression of mitosis. We propose that Bud6p, through its localization at both spindle pole bodies and at the mother-bud neck, supports this positive signal and provides a regulatory mechanism to prevent excessive oscillations of preanaphase nuclei, thus reducing the likelihood of mitotic delays and nuclear missegregation

A Genetic Dissection of Aip1p's Interactions Leads to a Model for Aip1p-Cofilin Cooperative Activities

Actin interacting protein 1 (Aip1p) and cofilin cooperate to disassemble actin filaments in vitro and are thought to promote rapid turnover of actin networks in vivo. The precise method by which Aip1p participates in these activities has not been defined, although severing and barbed-end capping of actin filaments have been proposed. To better describe the mechanisms and biological consequences of Aip1p activities, we undertook an extensive mutagenesis of AIP1 aimed at disrupting and mapping Aip1p interactions. Site-directed mutagenesis suggested that Aip1p has two actin binding sites, the primary actin binding site lies on the edge of its N-terminal β-propeller and a secondary actin binding site lies in a comparable location on its C-terminal β-propeller. Random mutagenesis followed by screening for separation of function mutants led to the identification of several mutants specifically defective for interacting with cofilin but still able to interact with actin. These mutants suggested that cofilin binds across the cleft between the two propeller domains, leaving the actin binding sites exposed and flanking the cofilin binding site. Biochemical, genetic, and cell biological analyses confirmed that the actin binding- and cofilin binding-specific mutants are functionally defective, whereas the genetic analyses further suggested a role for Aip1p in an early, internalization step of endocytosis. A complementary, unbiased molecular modeling approach was used to derive putative structures for the Aip1p-cofilin complex, the most stable of which is completely consistent with the mutagenesis data. We theorize that Aip1p-severing activity may involve simultaneous binding to two actin subunits with cofilin wedged between the two actin binding sites of the N- and C-terminal propeller domains