MOLECULAR-CLONING AND CELLULAR-LOCALIZATION OF A BIP HOMOLOG IN TRYPANOSOMA-BRUCEI - DIVERGENT ER RETENTION SIGNALS IN A LOWER EUKARYOTE

Using the polymerase chain reaction with degenerate primers, three new members of the hsp70 gene family of Trypanosoma brucei have been iden tified. A genomic clone of one of these, gA, has been fully sequenced and the corresponding gene product has been characterized using antibo dy to recombinant gA fusion protein. gA is the trypanosomal homologue of BiP, an endoplasmic reticulum resident hsp70 gene family member, ba sed on four lines of evidence: (1) gA protein has 64% deduced amino ac id identity with rat BiP; (2) the deduced amino acid sequence has a pu tative secretory signal peptide; (3) the gA gene product is a soluble luminal resident of a trypanosomal microsome fraction; (4) the gA poly peptide does not cofractionate with mitochondrial markers. Trypanosome s are the most primitive eukaryote yet in which BiP has been identifie d. The gA polypeptide has been used as a specific marker for the direc t visualization of endoplasmic reticulum in trypanosomes by both indir ect immunofluorescence and cryoimmuno electron microscopy. The endopla smic reticulum is seen as a tubular network that extends throughout th e cell excluding the flagellum. The C-terminal tetrapeptide of gA is M DDL, which, together with the C-terminal tetrapeptide (KQDL) of a tryp anosome protein disulfide isomerase homologue (Hsu et al. (1989) Bioch emistry 28, 6440-6446), indicates that endoplasmic reticulum retrieval signals in trypanosomes may be as divergent and heterogeneous as any seen in the other eukaryotes yet studied.