Suppression of Mutations in Two Saccharomyces cerevisiae Genes by the Adenovirus E1A Protein

Abstract

The protein products of the adenoviral E1A gene are implicated in a variety of transcriptional and cell cycle events, involving interactions with several proteins present in human cells, including parts of the transcriptional machinery and negative regulators of cell division such as the Rb gene product and p107. To determine if there are functional homologs of E1A in Saccharomyces cerevisiae, we have developed a genetic screen for mutants that depend on E1A for growth. The screen is based on a colony color sectoring assay which allows the identification of mutants dependent on the maintenance and expression of an E1A-containing plasmid. Using this screen, we have isolated five mutants that depend on expression of the 12S or 13S cDNA of E1A for growth. A plasmid shuffle assay confirms that the plasmid-dependent phenotype is due to the presence of either the 12S or the 13S E1A cDNA and that both forms of E1A rescue growth of all mutants equally well. The five mutants fall into two classes that were named web1 and web2 (for ''wants E1A badly''). Plasmid shuffle assays with mutant forms of E1A show that conserved region 1 (CR1) is required for rescue of the growth of the web1 and web2 E1A-dependent yeast mutants, while the N-terminal 22 amino acids are only partially required; conserved region 2 (CR2) and the C terminus are dispensable. The phenotypes of mutants in both the web1 and the web2 groups are due to a single gene defect, and the yeast genes that fully complement the mutant phenotypes of both groups were cloned. The WEB1 gene sequence encodes a 1,273-amino-acid protein that is identical to SEC31, a protein involved in the budding of transport vesicles from the endoplasmic reticulum. The WEB2 gene encodes a 1,522-amino-acid protein with homology to nucleic acid-dependent ATPases. Deletion of either WEB1 or WEB2 is lethal. Expression of E1A is not able to rescue the lethality of either the web1 or the web2 null allele, implying allele-specific mutations that lead to E1A dependence.

A surprisingly large RNase P RNA in Candida glabrata

“…Culture medium reagents were Fisher Scientific or Difco. The yeast strains used in this study were S. cerevisiae strain YMW1 (Zieler et al 1995) and C. glabrata strain Q (Zhou et al 1992).…”

Human Smp3p Adds a Fourth Mannose to Yeast and Human Glycosylphosphatidylinositol Precursors in Vivo

“…Plasmid p425-PGKhSMP3-HA was created by subcloning a SacI-XhoI fragment containing the PGK1 promoter and hSMP3-HA from p416-PGK-hSMP3-HA into the SacI-XhoI sites of pRS425. Plasmid pGAL-hSMP3-HA, in which expression of hSMP3-HA is controlled by the glucose-repressible GAL10 promoter, was made by cloning hSMP3-HA into pMW20 (34). Plasmids for yeast expression of ScSMP3 were constructed as follows.…”

Sec31 encodes an essential component of the COPII coat required for transport vesicle budding from the endoplasmic reticulum.

“…The carboxyl-terminal half of the protein is rich in serine, threonine, and proline residues but displays no significant homologies. During the characterization of this clone, we discovered that the gene encoding p150 is identical to WEBI, a locus defined by a single mutation webl-1 producing cells that require the viral protein ElA for growth in rich medium (Zieler et al, 1995). The DNA sequence is available from GenBank accession number U15219 (Zieler et al, 1995).…”