Characterization of the geranylgeranyl transferase type I from Schizosaccharomyces pombe.
The Schizosaccharomyces pombe cwg2+ gene encodes the beta-subunit of geranylgeranyl transferase I (GGTase I), which participates in the post-translational C-terminal modification of several small GTPases, allowing their targeting to the membrane. Using the two-hybrid system, we have identified the cwp1+ gene that encodes the alpha-subunit of the GGTase I. cwp1p interaction with cwg2p was mapped to amino acids 1-244 or 137-294 but was not restricted to amino acids 137-244. The genomic cwp1+ was isolated and sequenced. It has two putative open reading frames of 677 and 218 bp, separated by a 51 bp intron. The predicted amino acid sequence shows significant similarity to GGTase I alpha-subunits from different species. However, complementation of Saccharomyces cerevisiae ram2-1 mutant by overexpressing the cwp1+ gene was not possible. Expression of both cwg2+ and cwp1+ in Escherichia coli allowed 'in vitro' reconstitution of the GGTase I activity. S. pombe cells expressing the mutant enzyme containing the cwg2-1 mutation do not grow at 37 degrees C, but the growth defect can be suppressed by the addition of sorbitol. Actin immunostaining of the cwg2-1 mutant strain grown at 37 degrees C showed an abnormal distribution of actin patches. The cwg2-1 mutation was identified as a guanine to adenine substitution at nucleotide 604 of the coding region, originating the change A202T in the cwg2p. Deletion of the cwg2 gene is lethal; delta cwg2 spores can divide two or three times before losing viability. Most cells have aberrant morphology and septation defects. Overexpression of the rho1G15VC199R double-mutant allele in S. pombe caused loss of polarity but was not lethal and did not render the (1-3)beta-D-glucan synthase activity independent of GTP. Therefore, geranylgeranylation of rho1p is required for the appropriate function of this GTPase.
Arellano
M
M
Instituto de Microbiologia Bioquimica, CSIC/Universidad de Salamanca, Edificio Departamental, Spain.
Coll
P M
PM
Yang
W
W
Duran
A
A
Tamanoi
F
F
Perez
P
P
D000199:Actins / Q000378:metabolism; D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D005800:Genes, Fungal; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D017368:Protein Prenylation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D020741:rho GTP-Binding Proteins
D000199:Actins; D004271:DNA, Fungal; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; C511156:rho1 protein, S pombe