The Transcriptional Regulator Hap1p (Cyp1p) Is Essential for Anaerobic or Heme-Deficient Growth of Saccharomyces cerevisiae: Genetic and Molecular Characterization of an Extragenic Suppressor that Encodes a WD Repeat Protein

Abstract

We report here that Hap1p (originally named Cyp1p) has an essential function in anaerobic or heme-deficient growth. Analysis of intragenic revertants shows that this function depends on the amino acid preceding the first cysteine residue of the DNA-binding domain of Hap1p. Selection of recessive extragenic suppressors of a hap1−hem1− strain allowed the identification, cloning, and molecular analysis of ASC1 (Cyp1 Absence of growth Supressor). The sequence of ASC1 reveals that its ORF is interrupted by an intron that shelters the U24 snoRNA. Deletion of the intron, inactivation of the ORF, and molecular localization of the mutations show unambiguously that it is the protein and not the snoRNA that is involved in the suppressor phenotype. ASC1, which is constitutively transcribed, encodes an abundant, cytoplasmically localized 35-kD protein that belongs to the WD repeat family, which is found in a large variety of eucaryotic organisms. Polysome profile analysis supports the involvement of this protein in translation. We propose that the absence of functional Asc1p allows the growth of hap1−hem1− cells by reducing the efficiency of translation. Based on sequence comparisons, we discuss the possibility that the protein intervenes in a kinase-dependent signal transduction pathway involved in this last function.

Analysis of the Asc1p/RACK1 microenvironment in Saccharomyces cerevisiae using proximity-dependent Biotin Identification (BioID) and high-resolution mass spectrometry

“…by the transcription factors Fhl1p and Ifh1p that associate to many ribosomal gene promotors in S. cerevisiae (Kleinschmidt et al, 2006). In accordance with this, Asc1p comigrates with 40S ribosomal subunits in sucrose gradients, and the association of the β-propeller with 40S proteins is resistant to elevated salt concentrations as described for integral ribosomal proteins (Chantrel et al, 1998;Inada et al, 2002;Link et al, 1999). Asc1p is not present at the 40S pre-ribosome, which is exported from the nucleus into the cytoplasm for maturation, but is bound to the 80S-like ribosome, a non-translating intermediate during the final steps of 40S maturation (Strunk et al, 2012).…”

“…Thus, as a scaffold protein, Asc1p can be considered as a contact site of the ribosome for further cellular constituents. Immunofluorescence and centrifugation studies localized the Asc1 protein to the cytoplasm and to the cytosolic part of membranes (Baum et al, 2004;Chantrel et al, 1998). In contrast to its ribosome-associated localization during exponential growth, a ribosome-free cytosolic localization of an Asc1p sub-population was suggested in stationary yeast cells (Baum et al, 2004).…”

“…The Gβ-like Asc1 protein of S. cerevisiae belongs to the family of WD40 proteins and consists of seven WD40 repeats (Fig. 2A; Chantrel et al, 1998). Crystallization of recombinant S. cerevisiae Asc1p expressed in Escherichia coli revealed one single WD40 domain that folds into a seven-bladed β-propeller, the most common WD40 architecture characteristic for Gβlike proteins (Coyle et al, 2009;Stirnimann et al, 2010).…”

“…The 319 amino acids of the highly conserved Asc1 protein are encoded by a nucleotide sequence of 957 base pairs situated on two exons (Chantrel et al, 1998). The ASC1 gene is located on chromosome XIII and covers in total 1230 base pairs.…”

“…The ASC1 gene is located on chromosome XIII and covers in total 1230 base pairs. An intron of 273 base pairs bearing the SNR24 gene interrupts the ASC1 coding sequence at position 538 (Chantrel et al, 1998). The SNR24 gene encodes the small nucleolar RNA (snoRNA) U24, which is involved in nucleolar ribosome maturation (Kiss-László et al, 1996;Qu et al, 1995).…”