A small GTP-binding protein from Arabidopsis thaliana functionally complements the yeast YPT6 null mutant.
A clone designated A.t.RAB6 encoding a small GTP-binding protein was isolated from a cDNA library of Arabidopsis thaliana leaf tissue. The predicted amino acid sequence was highly homologous to the mammalian and yeast counterparts, H.Rab6 and Ryh1/Ypt6, respectively. Lesser homology was found between the predicted Arabidopsis protein sequence and two small GTP-binding proteins isolated from plant species (44% homology to Zea mays Ypt1 and 43% homology to Nicotiana tabacum Rab5). Conserved stretches in the deduced amino acid sequence of A.t.Rab6 include four regions involved in GTP-binding, an effector region, and C-terminal cysteine residues required for prenylation and subsequent membrane attachment. Northern blot analysis demonstrated that A.t.Rab6 mRNA was expressed in root, leaf, stem, and flower tissues from A. thaliana with the highest levels present in roots. Escherichia coli produced histidine-tagged A.t.Rab6 protein-bound GTP, whereas a mutation in one of the guanine nucleotide-binding sites (asparagine122 to isoleucine) rendered it incapable of binding GTP. Functionally, the A.t.RAB6 gene was able to complement the temperature-sensitive phenotype of the YPT6 null mutant in yeast. The isolation of this gene will aid in the dissection of the machinery involved in soluble protein sorting at the trans-Golgi network of plants.
Bednarek
S Y
SY
Department of Energy-Plant Research Laboratory, Michigan State University, East Lansing 48824-1312.
Reynolds
T L
TL
Schroeder
M
M
Grabowski
R
R
Hengst
L
L
Gallwitz
D
D
Raikhel
N V
NV
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D015152:Blotting, Northern; D015139:Blotting, Southern; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins; D009154:Mutation; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D019204:GTP-Binding Proteins; D020559:Monomeric GTP-Binding Proteins; C499307:YPT6 protein, S cerevisiae