Suppressor analysis of fimbrin (Sac6p) overexpression in yeast.
Yeast fimbrin (Sac6p) is an actin filament-bundling protein that is lethal when overexpressed. To identify the basis for this lethality, we sought mutations that can suppress it. A total of 1326 suppressor mutations were isolated and analyzed. As the vast majority of mutations were expected to simply decrease the expression of Sac6p to tolerable levels, a rapid screen was devised to eliminate these mutations. A total of 1324 mutations were found to suppress by reducing levels of Sac6p in the cell. The remaining 2 mutations were both found to be in the actin gene and to make the novel changes G48V (act1-20) and K50E (act1-21). These mutations suppress the defect in cytoskeletal organization and cell morphology seen in ACT1 cells that overexpress SAC6. These findings indicate that the lethal phenotype caused by Sac6p overexpression is mediated through interaction with actin. Moreover, the altered residues lie in the region of actin previously implicated in the binding of Sac6p, and they result in a reduced affinity of actin for Sac6p. These results indicate that the two mutations most likely suppress by reducing the affinity of actin for Sac6p in vivo. This study suggests it should be possible to use this type of suppressor analysis to identify other pairs of physically interacting proteins and suggests that it may be possible to identify sites where such proteins interact with each other.
Sandrock
T M
TM
Department of Molecular and Cellular Biology, Life Sciences South, University of Arizona, Tucson, Arizona 85721, USA.
Brower
S M
SM
Toenjes
K A
KA
Adams
A E
AE
D000199:Actins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000483:Alleles; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D016681:Genome, Fungal; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008840:Microfilament Proteins; D008958:Models, Molecular; D009154:Mutation; D010641:Phenotype; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D013489:Suppression, Genetic
D000199:Actins; D017931:DNA Primers; D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; C026742:plastin