Actin overexpression studies in Saccharomyces cerevisiae

Overexpression of actin is lethal in yeast. The toxicity of actin overexpression was exploited to discover new actin-related processes and actin-associated genes. Actin-overexpressing yeast cells were examined for alterations in gene expression patterns and for morphological defects. Genes involved in protein folding and degradation, as well as genes in the Environmental Stress Response were induced in actin-overexpressing cells. Fluorescence microscopy, indirect immuno-fluorescence microscopy, and immuno-electron microscopy reveal that cells grow abnormally large, accumulate secretory vesicles, and have incomplete septa and cell wall defects. In addition, they display aberrant filamentous actin structures, including rings of actin within the cell nucleus.;A genetic screen was performed to identify novel actin-associated genes, by selecting for suppression of the lethality of actin overexpression. Mutations in four different genes were recovered which allow yeast to survive actin overexpression. A mutation in the endogenous actin gene may confer resistance to overexpressed actin by lowering the total amount of actin in the cell. Another mutation in the gene encoding the beta-subunit of the actin capping protein may confer resistance by destabilizing filamentous actin. This suggests that f-actin, not monomeric actin, is the cause of the lethality of actin overexpression.;A yeast genomic DNA library was constructed to isolate clones which complement the remaining mutations. This library contains a drug resistance marker, allowing it to be used in situations were auxotrophic markers are not available, or when selecting phenotypes dependent upon rich media.;One of the complementing clones encodes a previously uncharacterized gene called AOR1 (for Actin Overexpression Resistant). Aor1 protein contains a C-terminal domain conserved in all eukaryotes. An Aor1 protein tagged with Green Fluorescent Protein localizes to the nucleus. In addition to conferring resistance to actin overexpression, null mutations in AOR1 confer moderate cold sensitivity, confer hypersensitivity to the microtubule-destabilizing drug benomyl, and suppress the formation of f-actin structures in the nuclei of actin-overexpressing cells. Genetic tests suggest that AOR1 interacts with genes involved in f-actin assembly, mitotic spindle function, nuclear trafficking, and nuclear positioning. AOR1 may therefore provide a link between the actin and tubulin cytoskeletons in these diverse processes.