Characterization of a Phytophthora infestans gene involved in vesicle transport

Abstract

Members of the Ras superfamily of monomeric GTP-binding proteins have been shown to be essential in specific steps of vesicle transport and secretion in widely divergent organisms. We report here the characterization of a gene from Phytophthora infestans encoding a deduced amino acid (aa) sequence belonging to the Ypt class of monomeric GTP-binding proteins, products shown in other organisms to be essential for vesicle transport between the endoplasmic reticulum and the cis-Golgi compartments. Analysis of genomic and cDNA sequences of this gene, Piyptl, indicates that it contains five introns, one in the 5′-untranslated region. All introns are typical in beginning with GT and ending with AG. The region of the transcription start point displays a number of features characteristic of fungi and other eukaryotes, but it does not contain TATA or CAAT motifs. A single transcript is produced from the gene, which is polyadenylated, but the gene does not contain a recognizable polyadenylation signal. Genomic DNA blots indicate that Piyptl is a single-copy gene. Comparisons of Yptl aa sequences indicate that P. infestans is more closely related to algae and higher plants than to the true fungi. The protein product of the Piyptl gene, expressed in Escherichia coli, cross-reacts with antiserum against yeast Yptl protein and binds GTP. Furthermore, the Piyptl gene is able to functionally complement a mutant ypt1 gene in Saccharomyces cerevisiae. The aa sequence similarity, immunological cross-reactivity and functional attributes of Piyptl make it likely that it is an authentic yptl gene which participates in vesicle transport in Phytophthora infestans