Jd. Bangs et al., MOLECULAR-CLONING AND CELLULAR-LOCALIZATION OF A BIP HOMOLOG IN TRYPANOSOMA-BRUCEI - DIVERGENT ER RETENTION SIGNALS IN A LOWER EUKARYOTE, Journal of Cell Science, 105, 1993, pp. 1101-1113
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.