In the budding yeast Kluyveromyces marxianus , adenylate cyclase is regulated by Ras protein(s) in vitro

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.

Section: Resultsmentioning

confidence: 99%

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

Saviñón‐Tejeda

Ongay‐Larios

Ramı́rez

et al. 1996

“…The present study concerns the induction, purification and characterization of an amine oxidase (KMAO) from the yeast Kluyveromyces marxianus and its partial cDNA sequencing. This microorganism, phylogenetically close to Saccharomyces cerevisiae , has already been studied in order to elucidate its metabolism, biochemistry and genetics (Verzotti, 1994; Siekstele, 1999; Ladrière, 2000; Llorente, 2000; Belloch, 2002; Pinheiro, 2002), for applications (Medeiros, 2000; Meehan, 2000; Kourkoutas, 2002) and especially for its great resistance to high concentrations of metals and its ability to absorb and then to release them (Yazgan, 1993; Aksu, 2000).…”

Section: Introductionmentioning

confidence: 99%

Induction and characterization of a novel amine oxidase from the yeast Kluyveromyces marxianus

Corpillo¹,

Valetti²,

Giuffrida³

et al. 2003

Self Cite

An amine oxidase from the yeast Kluyveromyces marxianus was induced, purified and completely characterized; it was shown to belong to the class of coppercontaining amine oxidases (E.C. 1.4.3.6). The enzyme was induced by putrescine and, very strongly, by copper(II); structural-functional characterization of the enzyme was performed, including determination of molecular weight, glycosylation, copper and TPQ content, isoelectric point, K M and k CAT (with benzylamine as substrate), pH, temperature and ionic strength effect on catalysis, substrate and inhibitor specificity. A 700 bp clone was isolated containing the cDNA that encodes for the C-terminus of the enzyme; the amino acid sequence deduced (the first available for a benzylamine oxidase from yeast) was compared to that of other copper amine oxidases from microorganisms and higher organisms. From the results obtained, the putrescine/benzylamine oxidase from Kluyveromyces marxianus was found to have a good homology with other enzymes of this class from microorganisms, and particularly with AO I from Aspergillus niger. Nonetheless, some features resulted closer to those of animal amine oxidases and histaminases. Some potential biotechnological applications are proposed. The cDNA Accession No. is AJ320485.

Isolation of a gene encoding a G protein α subunit involved in the regulation of cAMP levels in the yeastKluyveromyces lactis

Saviñón‐Tejeda

Ongay‐Larios

Ramı́rez

et al. 1996

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 Gα 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 α subunits from Saccharomyces cerevisiae. KlGPA2 is a single‐copy gene and its disruption rendered viable cells with significantly reduced cAMP level, indicating that this Gα 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 Gαs family (stimulatory of adenylyl cyclase) including the absence in its N‐terminus of a myristoyl‐modification sequence. The sequence reported in this paper has been deposited in the GenBank data base (Accession No. L45105).