BAP, a Mammalian BiP-associated Protein, Is a Nucleotide Exchange Factor That Regulates the ATPase Activity of BiP

Abstract

We identified a mammalian BiP-associated protein, BAP, using a yeast two-hybrid screen that shared low homology with yeast Sls1p/Sil1p and mammalian HspBP1, both of which regulate the ATPase activity of their Hsp70 partner. BAP encoded an ϳ54-kDa protein with an N-terminal endoplasmic reticulum (ER) targeting sequence, two sites of N-linked glycosylation, and a C-terminal ER retention sequence. Immunofluorescence staining demonstrated that BAP co-localized with GRP94 in the endoplasmic reticulum. BAP was ubiquitously expressed but showed the highest levels of expression in secretory organ tissues, a pattern similar to that observed with BiP. BAP binding was affected by the conformation of the ATPase domain of BiP based on in vivo binding studies with BiP mutants. BAP stimulated the ATPase activity of BiP when added alone or together with the ER DnaJ protein, ERdj4, by promoting the release of ADP from BiP. Together, these data demonstrate that BAP serves as a nucleotide exchange factor for BiP and provide insights into the mechanisms that control protein folding in the mammalian ER.

Novel SIL1 mutations and exclusion of functional candidate genes in Marinesco–Sjögren syndrome

“…We first tested the localization of wild-type SIL1. Contrary to the previous studies that have reported ER localization, 8,11 we detected SIL1 in both the ER and the Golgi apparatus. We then investigated the subcellular localization of endogenous SIL1 in mouse hippocampal neurons, where we previously showed SIL1 expression.…”

“…11 SIL1 is a 461 aminoacid protein that has a potential N-terminal ER targeting sequence and a C-terminal tetrapeptide (KELR; amino acids 458 -461), quite likely to be an ER retrieval sequence. 8 In previous studies, transiently expressed SIL1 has been shown to localize in the ER. 8,11 The tissue expression pattern of SIL1 is similar to that of GRP78 in mouse, thereby supporting their functional interaction.…”

“…8 In previous studies, transiently expressed SIL1 has been shown to localize in the ER. 8,11 The tissue expression pattern of SIL1 is similar to that of GRP78 in mouse, thereby supporting their functional interaction. 4 The cerebellar atrophy seen in patients with MSS is caused by loss of Purkinje and granule cells.…”

“…5 SIL1 acts as an adenine nucleotide exchange factor for the heat-shock protein 70 (Hsp70) chaperone GRP78 (also known as BiP and HSPA5), a molecular chaperone functioning mainly in the endoplasmic reticulum (ER). 8,9 SIL1 regulates the ATPase cycle of GRP78 and has thus been anticipated to be involved in protein translocation into the ER, proper folding of the newly synthesized proteins, regulating the degradation of proteins that fail to mature properly, and responding to cell stress. 10 However, recent data imply that SIL1 is most likely not involved in the regulation of the unfolded protein response (UPR), as the UPR is upregulated normally in the woozy (wz) mouse, a spontaneous Sil1 mouse mutant.…”

The Mammalian Hsp40 ERdj3 Requires Its Hsp70 Interaction and Substrate-binding Properties to Complement Various Yeast Hsp40-dependent Functions

“…For immunofluorescence (22), transfected cells grown on coverslips were fixed and stained with an anti-HA antibody to detect ssYdj1 followed by fluorescein isothiocyanate-labeled secondary antibody. Grp94, an abundant ER lumenal protein, served as an ER marker and was detected with an anti-Grp94 antiserum (22) followed by a TRITC-conjugated secondary antibody.…”