Analysis of a family of ypt genes and their products from Chlamydomonas reinhardtii

Abstract

Small G-proteins encoded by the ras-like ypt genes are ubiquitous in eukaryotic cells. They have been shown to play an essential role in membrane vesicle transport. We have isolated four ypt genes, yptC1, yptC4, yptC5 and yptC6, from Chlamydomonas reinhardtii (Cr) genomic and cDNA libraries. Three of them, yptC1, yptC4 and yptC5, are close homologues of ypt genes previously found in the multicellular alga Volvox carteri (Vc), the fourth, yptC6, is new. Each yptC gene is present as a single copy in the genome. Comparisons of genomic and cDNA sequences revealed that the coding regions are interrupted by five (yptC5), six (yptC6), seven (yptC4) and eight (yptC1) introns, respectively. Cr ypt genes and the closely related Vc ypt genes have identical exon-intron structures, but the corresponding intron sequences are completely different. Polyadenylation is signalled by UAUAA, UGUAG and UGUAA. The deduced amino acid (aa) sequence of YptC6 exhibited 79% identity with HRab2; YptC1, YptC4 and YptC5 exhibited over 90% identity with their Vc homologues. Primary structures of the 9-aa 'effector domain' and the contiguous 'helix3-loop7' motif (approx. 30 aa) are 'diagnostic' features for functional assignment. Recombinant YptC proteins, overproduced in Escherichia coli and purified to near homogeneity, displayed strong and specific binding of GTP, but not of GMP or ATP. The four Cr Ypt proteins showed immunochemical cross reactions to their Vc counterparts. Moreover, Western blots demonstrated at least six types of Ypt in both Cr and Vc, suggesting that these Ypt are used for household functions responsible for vesicle transport rather than for cellular differentiation.