Isolation of a Gene Encoding a G Protein α Subunit Involved in the Regulation of cAMP Levels in the Yeast Kluyveromyces lactis

Abstract

Using chromosomal DNA from Kluyveromyces lactis as template and oligodeoxynucleotides designed from conserved regions of various G protein alpha subunits we were able to amplify by the polymerase chain reaction two products of approximately 0.5 kb (P-1) and 0.8 kb (P-2). Sequencing showed that these two fragments share high homology with genes coding for the G alpha subunits from different sources. Using the P-1 fragment as a probe we screened a genomic library from K. lactis and we cloned a gene (KlGPA2) whose deduced amino acid sequence showed, depending on the exact alignment, 62% similarity and 38% identity with Gpa1p and 76% similarity and 63% identity with Gpa2p, the G protein alpha subunits from Saccharomyces cerevisiae. KlGPA2 is a single-copy gene and its disruption rendered viable cells with significantly reduced cAMP level, indicating that this G alpha subunit may be involved in regulating the adenylyl cyclase activity, rather than participating in the mating pheromone response pathway. KlGpa2p shares some structural similarities with members of the mammalian G alpha s family (stimulatory of adenylyl cyclase) including the absence in its N-terminus of a myristoyl-modification sequence.

The KlGpa1 Gene Encodes a G-Protein α Subunit That Is a Positive Control Element in the Mating Pathway of the Budding Yeast Kluyveromyces lactis

“…lactis has at least two G-protein ␣ subunits that are involved in two different pathways. While KlGpa1p participates in the mating pathway, KlGpa2p is implicated in regulation of the adenylyl cyclase (25) and, as we showed here, is able to partially interfere with mating. These data suggest cross talk between these two proteins and, although we have not investigated a possible role of KlGpa1p in cAMP regulation, a relationship between cAMP and mating signaling pathways may exist.…”

“…We have already demonstrated that one of these genes, KlGpa2, encodes a subunit directly involved in the regulation of cAMP levels in K. lactis (25). Cells lacking KlGpa2p are viable but fail to respond to transient stimulus by glucose and the cAMP level drops significantly, indicating that the G protein containing KlGpa2p is involved in regulating the activity of adenylyl cyclase and participates in a pathway related to monitoring the nutrient status of the cell (25).…”

“…This cassette was used to transfect haploid and diploid strains. Disruption of the KlGpa2 allele with the Ura3 marker was described previously (25). Disruption of KlGpa2 with the His3 cassette was as follows.…”

“…In particular, here we describe the characterization of a gene encoding a G␣ subunit that participates in the mating pathway. Like S. cerevisiae, K. lactis expresses a second G␣ subunit (Gpa2p) that is directly involved in regulation of cAMP levels in response to glucose stimulus (25).…”

Sex and Crime: Heterotrimeric G Proteins in Fungal Mating and Pathogenesis

“…A similar function had been previously observed for G␣ proteins in Schizosaccharomyces pombe (Isshiki et al, 1992) and Kluyveromyces lactis (Saviñ on-Tejeda et al, 1996).…”