Structure-function analysis of small G proteins from Volvox and Chlamydomonas by complementation of Saccharomyces cerevisiae YPT/SEC mutations.
cDNAs representing nine small G protein genes encoding Ypt proteins from the green algae Volvox carteri (YptV) and Chlamydomonas reinhardtii (YptC) were tested for their ability to complement mutations in the YPT1, SEC4, and YPT7 genes of Saccharomyces cerevisiae strains defective in different steps of intracellular vesicle transport. None of the heterologously expressed algal genes was able to complement mutations in SEC4 or YPT7, but three of them, yptV1, yptC1, and yptV2, restored a YPT1 null mutation. On the amino acid sequence level, and particularly with respect to known small G protein specificity domains, YptV1p and YptC1p are the closest algal analogs of yeast Ypt1p, with 70% overall identity and identical effector regions, but YptV2p is only 55% identical to Ypt1p, and its effector domain resembles that of Sec4p. To define more precisely the regions that supply Ypt1p function, six chimeras were constructed by reciprocal exchange of 68/72-, 122/123-, and 162/163-amino acid segments of the C-terminal regions between YptV1p (complementing) and YptV3p (non-complementing). Segments containing 68 amino acids of the hypervariable C-terminal, and 41 residues of the N-terminal region including the effector region, of YptV1p could be replaced by the corresponding parts of YptV3p without loss of function in yeast, but exchanges within the central core destroyed the ability to rescue the YPT1 mutation. Sequence analysis of ypt1-complementing and -noncomplementing Ypt types suggests that surface loop3 represents a novel specificity domain of small G proteins.
Fabry
S
S
Universität Regensburg, Institut für Biochemie, Genetik und Mikrobiologie, Lehrstuhl für Genetik, Germany.
Steigerwald
R
R
Bernklau
C
C
Dietmaier
W
W
Schmitt
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics* / Q000378:metabolism; D000460:Chlorophyta / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D005656:Fungal Proteins / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D020691:rab GTP-Binding Proteins
D017931:DNA Primers; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D019204:GTP-Binding Proteins; C060755:YPT1 protein, S cerevisiae; C064320:SEC4 protein, S cerevisiae; D020691:rab GTP-Binding Proteins