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 po st-translational C-terminal modification of several small GTPases, all owing their targeting to the membrane. Using the two-hybrid system, we have identified the cwp1(+) gene that encodes the alpha-subunit of th e GGTase I. cwp1(+) interaction with cwg2p was mapped to amino acids 1 -244 or 137-294 but was not restricted to amino acids 137-244. The gen omic cwp1(+) was isolated and sequenced. It has two putative open read ing frames of 677 and 218 bp, separated by a 51 bp intron. The predict ed amino acid sequence shows significant similarity to GGTase I alpha- subunits from different species. However, complementation of Saccharom yces 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 c ells expressing the mutant enzyme containing the cwg2-1 mutation do no t grow at 37 degrees C, but the growth defect can be suppressed by the addition of sorbitol. Actin immunostaining of the cwg2-1 mutant strai n grown at 37 degrees C showed an abnormal distribution of actin patch es. The cwg2-1 mutation was identified as a guanine to adenine substit ution at nucleotide 604 of the coding region, originating the change A 202T in the cwg2p. Deletion of the cwg2 gene is lethal; Delta cwg2 spo res 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.