A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum.

Abstract

Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates. Photoaffinity labelling of microsomes with azido‐ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61alpha and TRAM proteins. The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP‐binding proteins by passage through ATP‐agarose and subsequent reconstitution of the pass‐through into proteoliposomes. Transport was restored by co‐reconstitution of the ATP eluate. This eluate showed eight distinct bands in SDS gels. We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5). In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP‐agarose. Purified BiP did not stimulate transport activity. Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes. We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates.

Targeting to Static Endosome Is Required for Efficient Cross-Presentation of Endoplasmic Reticulum-Resident Oxygen-Regulated Protein 150-Peptide Complexes

“…This may be because ORP150 is more efficient in binding peptides than is Hsp70. The ability of ORP150 to bind polypeptides better than other Hsp70 family members is largely due to its enlarged C-terminal helical domain (41,42). Moreover, it may help that peptide binding to ORP150 is independent of ATP, which is in contrast with ATP-dependent substrate binding of other Hsp70 family members (42).…”

Characterization of Heat Shock Protein 110 and Glucose-Regulated Protein 170 as Cancer Vaccines and the Effect of Fever-Range Hyperthermia on Vaccine Activity

“…Less is understood at the molecular level of grp170 structure and function; however, cellular studies have shown that it binds to Ig chain in the ER, may be the ATPase responsible for protein import into the ER, and actively binds peptides from TAP (i.e., the transporter associated with Ag processing; Refs. 11,19,20,23,24).…”

Conversion of cysteine to formylglycine: A protein modification in the endoplasmic reticulum

“…The system is based on SP6-or T7-polymerase, reticulocyte lysate, and on either [ 35 S]methionine or [ 35 S]cysteine (Amersham) and was programmed with circular DNA. Rough microsomes from dog pancreas (11) were added at 7.5 equivalents (12) per 50 l translation mixture. After incubation for 45 min at 30°C the microsomes were sedimented for 5 min at 75,000 ϫ g and treated with 170 g͞ml proteinase K for 45 min at 0°C.…”

“…In vitro translation products were chilled on ice and aliquots were boiled immediately with electrophoresis sample buffer. SDS͞ PAGE and fluorography were as described (11). High-Tris gels containing 17.5% acrylamide͞0.23% bis-acrylamide or (Fig.…”