Molecular cloning and cellular localization of a BiP homologue 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 identified. A genomic clone of one of these, gA, has been fully sequenced and the corresponding gene product has been characterized using antibody to recombinant gA fusion protein. gA is the trypanosomal homologue of BiP, an endoplasmic reticulum resident hsp70 gene family member, based on four lines of evidence: (1) gA protein has 64% deduced amino acid identity with rat BiP; (2) the deduced amino acid sequence has a putative secretory signal peptide; (3) the gA gene product is a soluble luminal resident of a trypanosomal microsome fraction; (4) the gA polypeptide does not cofractionate with mitochondrial markers. Trypanosomes are the most primitive eukaryote yet in which BiP has been identified. The gA polypeptide has been used as a specific marker for the direct visualization of endoplasmic reticulum in trypanosomes by both indirect immunofluorescence and cryoimmuno electron microscopy. The endoplasmic reticulum is seen as a tubular network that extends throughout the cell excluding the flagellum. The C-terminal tetrapeptide of gA is MDDL, which, together with the C-terminal tetrapeptide (KQDL) of a trypanosome protein disulfide isomerase homologue (Hsu et al. (1989) Biochemistry 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.
Bangs
J D
JD
Department of Microbiology and Immunology, Stanford School of Medicine, CA 94305-5402.
Uyetake
L
L
Brickman
M J
MJ
Balber
A E
AE
Boothroyd
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics; D017125:Genes, Protozoan; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006651:Histocytochemistry; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure
D016054:DNA, Protozoan; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C060153:KAR2 protein, yeast; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins