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Lethal Disruption of the Yeast Actin Gene by Integrative DNA Transformation

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Abstract

A mutant allele of the chromosomal locus corresponding to the cloned actin gene of the yeast Saccharomyces cerevisiae has been constructed by DNA transformation with a hybrid plasmid which integrates into, and thereby disrupts, the protein-encoding sequences of the gene. In a diploid strain of yeast, disruption of the actin gene on one chromosome results in a mutation that segregates as a recessive lethal tightly linked to a selectable genetic marker on the integrated plasmid. The actin gene, therefore, must encode an essential function for yeast cell growth.
... Mass spectrometry data showed that Vps1 and Act1 were obviously Nt acetylated by Nat3 ( Figure S3C). Knockout of Vps1 and disturbance of filament formation by Act1 (Act1 could not be knocked out since it is essential for cell growth) (Shortle et al., 1982) with the chemical latrunculin A (LatA) (Coué et al., 1987;Varland et al., 2019) or by promoter-shutoff expression of actin nucleation factor Arp2 (Mnaimneh et al., 2004;Winter et al., 1997) blocked autophagy (Figures 3B and 3C;Figures S3D and S3E). This indicated that Vps1 or actin filaments produced by Act1 are essentially involved in autophagy. ...
... Nt-acetylation of Act1 regulates its function in autophagosome formation We next tried to determine how Nt-acetylation of Act1 regulates its function in autophagy. Since Act1 could not be knocked out because it is essential for cell growth (Shortle et al., 1982), we used LatA treatment or promoter-shutoff expression of actin nucleation factor of Arp2 to disrupt the actin filaments formed by Act1 ( Figure S3E). This caused the blockage of autophagic A B C D Figure 2. Acetyltransferase activity of Nat3 is essential for its function in autophagy (A) Schematic representation of yeast Nat3 protein and indicated truncates that were checked for function in autophagy. ...
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