Guanine nucleotide-binding proteins in the intestinal parasite Giardia lamblia. Isolation of a gene encoding an approximately 20-kDa ADP-ribosylation factor

Abstract

Giardia lamblia is a protozoan intestinal parasite that has characteristics of both eukaryotes and prokaryotes. To determine whether genes for guanine nucleotide-binding proteins are present in Giardia, genomic DNA and cDNA libraries were screened by polymerase chain reaction and by hybridization with mixed oligonucleotide probes complementary to sequences encoding conserved GTP-binding domains. A gene with a high degree of sequence identity with mammalian ADP-ribosylation factors (ARFs), believed to be important in vesicular transport, was identified. The Giardia ARF gene had a 573-base open reading frame encoding 191 amino acids which are 63-70% identical with known mammalian and yeast ARFs. Sequence conservation among ARFs was greatest in putative GTP-binding domains. A single ARF mRNA species of approximately 750 bases was found in two different Giardia isolates. Primer extension and RNA sequencing of the Giardia ARF transcript revealed a short (6-base) 5'-untranslated region similar in size to those found in other Giardia transcripts. Giardia extracts contained ARF activity, as shown by stimulation of cholera toxin-catalyzed ADP-ribosylation and a Giardia ARF expressed in Escherichia coli as a fusion protein likewise exhibited biochemical activity. Its presence in Giardia is consistent with the view that ARF emerged before the divergence of this protozoan from other eukaryotes (approximately 1.5 billion years ago), and that an ARF-like protein may have been the ancestor of several other classes of signal-transducing guanine nucleotide-binding proteins, including the alpha subunits of the heterotrimeric G proteins.