Similarity of nucleotide interactions of BiP and GTP-binding proteins.

Abstract

BiP is a member of the Hsp7O heat shock protein family found in the lumen of the endoplasmic reticulum, that binds to a variety of proteins destined to be secreted. Substance P (SP) has been used as a model peptide to study the interaction of BiP with protein substrates. SP stimulates BiP ATPase activity and forms a stable complex with BiP that is dissociated in the presence of levels of ATP > 50 pM. At lower concentrations of ATP, the SP remains bound to BiP, and the results are consistent with the view that a BiP-ATP complex is initially formed that reacts with SP to form a ternary complex, SP-BiP-ATP. Hydrolysis of ATP in this complex yields a SP-BiP-ADP complex. An exchange of ATP with ADP bound to BiP has also been demonstrated, and the results suggest that the interactions of BiP with ATP resemble those seen with GTP-binding proteins and GTP.Heat shock proteins (Hsps) or stress proteins, such as members of the Hsp7O family, interact with proteins to facilitate proper folding and assembly within the cell (1-3). These proteins are known to interact with ATP (4, 5) and possess a week ATPase activity (6-9) that can be stimulated by peptides and proteins that bind to the chaperone (7,10,11). ATP binding and/or hydrolysis is also believed to be involved in the dissociation of the Hsp70-protein complex (1)(2)(3)(10)(11)(12)(13)(14)(15), and recently, several studies have examined the interaction of ATP, ADP, and polypeptides with members of the Hsp7O family (4,(10)(11)(12)(13)(14).One member of this group called BiP (immunoglobulin heavy chain binding protein) or Grp78 (78-kDa glucoseregulated protein) is found in the lumen of the endoplasmic reticulum (ER), where it interacts transiently with some, but not all, nascent polypeptides destined to be secreted (3,(16)(17)(18)(19)(20). However, BiP also forms much tighter complexes with malfolded proteins (e.g., mutated and underglycosylated proteins), unassembled polypeptides, etc. (21)(22)(23)(24). In these cases the BiP-protein complexes are so tight that transport through the ER is prevented. Previously we initiated experiments to investigate the interaction of ATP with BiP, using either BiP isolated from liver (IBiP) or a recombinant form (rBiP) made in Escherichia coli (9, 13). We showed, using gel-filtration chromatography, that ATP could dissociate BiP dimers to monomers and that ATP bound to the monomer species (9, 13). In addition, in cell extracts a BiP-IgE Fc complex that accumulates in the ER was dissociated by ATP to yield a BiP monomer (13 (11) proposed a similar scheme for the interaction of BiP with substrates. In the present study we show that the 11-amino acid peptide substance P (SP), which was reported to bind to BiP (27), acts as a model substrate for BiP interactions on the basis of criteria first described by Flynn et al. (7). A rapid filter-binding assay has been used to examine the effect of SP on nucleotide binding to BiP, and our results are consistent with an initial interaction of SP with a BiP-ATP complex and a subsequent series...

Effect of Constitutive 70-kDa Heat Shock Protein Polymerization on Its Interaction with Protein Substrate

“…In contrast to the in vivo study by Freiden et al (1992), two of these studies found evidence for peptide binding to polymerized BiP in vitro (Blond-Elguindi et al, 1993;Brot et al, 1994). Unfortunately, these two studies disagree with each other in major respects.…”

“…Blond-Elguindi et al (1993) found that neither recombinant BiP nor BiP prepared from liver were depolymerized at low concentrations of ATP, although peptide depolymerized BiP. On the other hand, Brot et al (1994) found that low concentrations of ATP depolymerizes BiP almost completely, although peptide binding had no effect on the amount of polymer.…”

“…However, recently there have been contradictory reports as to whether BiP occurs as monomers at low ATP concentration (BlondElguindi et al, 1993;Brot et al, 1994). Fig.…”

“…Since they obtained preliminary data showing that cytoplasmic hsc70 behaved similarly, they argued that this model might be general for all hsc70s. Brot et al (1994) also studied the interaction of BiP with substrates and ATP, and they too observed direct binding of a substrate (substance P) to BiP dimers in ADP. However, in direct contrast to the findings of Blond-Elguindi et al, they found that although ATP monomerized BiP, the binding of substance P did not.…”

A revisit to bacterial protein synthesis: The search for the role of GTP

“…One such example may be the interaction of chaperons, such as the hsp 70 family, with malfolded proteins and adenosine nucleotides. Although ATP and ADP are involved in these reactions, and not guanosine nucleotides, it is quite easy to formulate a series of reactions with similarities to those seen with G proteins (Brot et al, 1994).…”