In the present study, we produced single point mutations in the ATP binding site of hamster BiP, isolated recombinant proteins, and characterized them in terms of their affinity for ATP and ADP, their ability to undergo a conformational change upon nucleotide binding, and their rate of ATP hydrolysis. These analyses allowed us to classify the mutants into three groups: ATP hydrolysis (T229G), ATP binding (G226D, G227D), and ATP-induced conformation (T37G) mutants, and to test the role of these activities in the in vitro ATP-mediated release of proteins from BiP. All three classes of mutants were still able to bind peptide demonstrating that nucleotide is not involved in this function. Addition of ATP to either wild-type BiP or the T229G mutant caused the in vitro release of bound peptide, confirming that ATP hydrolysis is not required for protein release. ATP did not dissociate G226D, G227D, or T37G mutant BiP-peptide complexes, suggesting that ATP binding to BiP is not sufficient for the release of bound peptides, but that an ATP-induced conformational change in BiP is necessary. The identification of BiP mutants that are defective in each of these steps of ATP hydrolysis will allow the in vivo dissection of the role of nucleotide in BiP's activity.The heat shock protein 70 (HSP70) 1 family of chaperones are components of the cellular machinery for folding, assembly, and degradation of proteins (1). These proteins are thought to undergo cycles of nucleotide-mediated binding and release to unfolded polypeptides. The binding of peptides to HSP70 proteins stimulates their ATPase activity (2), and bound peptides or proteins are released with ATP but not with non-hydrolyzable analogues (2-5). These observations have led to the conclusion that ATP hydrolysis is required for HSP70 activity and that there are functional interactions between the ATP binding and protein binding domains. All HSP70 proteins bind ATP tightly but the ATP hydrolysis rates of purified proteins are so low under physiological conditions, that other co-factors may be required to enhance the rate of ATP hydrolysis. In bacteria two co-factors, dnaJ and grpE, have been identified that together increase the ATPase activity of dnaK (bacterial HSP70 homologue) up to 50-fold (6). Although dnaJ homologues have been identified in several organelles in various organisms (7), the only eukaryotic grpE homologues found thus far are mitrochondrial (8, 9). Alternatively, it is possible that ATP binding rather than ATP hydrolysis is essential for HSP70 function. In support of this hypothesis, investigators have recently demonstrated that peptides are released from both dnaK and hsc70 (mammalian cytosolic homologue) after ATP binding but before ATP hydrolysis occurs (10). Thus, it is presently unclear whether ATP binding, ATP hydrolysis, or both are important for HSP70 function in vivo.The ATP binding domain of all HSP70 members resides within a highly conserved NH 2 -terminal 44-kDa fragment that can be generated by proteolysis (11,12). The structure of the ATP ...

The immunoglobulin heavy chain binding protein BiP/GRP78 is post‐translationally modified by phosphorylation and ADP ribosylation. In cells induced to synthesize higher levels of BiP, either due to the accumulation of nontransported proteins or to glucose starvation, both BiP phosphorylation and ADP ribosylation are reduced. BiP bound to other proteins is unmodified, suggesting that both phosphorylation and ADP ribosylation are restricted to the unbound BiP pool. In the present study, both modifications were further characterized in terms of their stability, the pool of BiP that harbored these modifications, and the relationship between the modified and unmodified forms of BiP. While levels of BiP synthesis vary according to the physiological state of a cell, we found that both induced and uninduced cells contain similar amounts of free BiP. However, free BiP in uninduced cells was found primarily in an aggregated state, whereas in cells that accumulate nontransported proteins, it was predominantly monomeric. Both phosphorylation and ADP ribosylation were restricted to the aggregated form of free BiP. These post‐translational modifications occurred upon release of BiP from associated proteins, and could be reversed upon induction of BiP synthesis. Therefore, BiP exists either (1) complexed to other proteins, (2) as a free unmodified monomer, or (3) as free modified aggregates. Our data suggest that BiP can be interconverted from one state to another, and that the various forms are functionally distinct.

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Recent studies of cellular amyloid precursor protein (APP) metabolism demonstrate a ␤-/␥-secretase pathway resident to the endoplasmic reticulum (ER)/Golgi resulting in intracellular generation of soluble APP (APPs␤) and A␤42 peptide. Thus, these intracellular compartments may be key sites of amyloidogenic APP metabolism and Alzheimer's disease pathogenesis. We hypothesized that the ER chaperone immunoglobulin binding protein (BiP/GRP78) binds to and facilitates correct folding of nascent APP. Metabolic labeling and immunoprecipitation of transiently transfected human embryonic kidney 293 cells demonstrated co-precipitation of APP with GRP78, revealing their transient interaction in the ER. Maturation of cellular APP was impaired by this interaction. Furthermore, the levels of APPs, A␤40, and A␤42 recovered in conditioned medium were lower compared with cells transfected with APP alone. Coexpression with APP of GRP78 T37G, an ATPase mutant, almost completely blocked cellular APP maturation as well as recovery of APPs, A␤40, and A␤42 in conditioned medium. The inhibitory effects of GRP78 and GRP78 T37G on A␤40 and A␤42 secretion were magnified by co-expression with the Swedish mutation of APP (K670N/M671L). Collectively, these data suggest a transient and direct interaction of GRP78 with APP in the ER that modulates intracellular APP maturation and processing and may facilitate its correct folding.The major components of amyloid plaque in Alzheimer's disease (AD) 1 brain are A␤ peptides, including A␤40 and A␤42, that are derived from amyloid precursor protein (APP). APP is metabolized at or near the cell surface by an ␣-secretase that results in soluble APP (APPs␣) secretion and precludes A␤ formation. APP is also metabolized by an endosomal/lysosomal (endocytic) pathway that results in A␤ secretion (1-3). Recent data with human NT2 neurons demonstrates that A␤ is found intracellularly (4) with kinetics identical to APP synthesis, suggesting that a fraction of nascent APP is immediately metabolized to A␤ (5). Subsequently, a ␤-/␥-secretase pathway of APP metabolism resident to the endoplasmic reticulum (ER)/ Golgi of neurons was identified, resulting in intracellular APPs␤ and A␤42 generation (6 -11). This exocytic pathway may be specifically promoted by presenilin-1 and presenilin-2 mutations found in some pedigrees of familial AD, since these proteins localize to the ER/Golgi (12-14) and result in greater A␤42 secretion (15-17). In fact, common to all mutations of APP and presenilins linked to early-onset familial AD is their ability to promote A␤42 generation (1-3). Because A␤42 is intrinsically more amyloidogenic than A␤40 and deposits preferentially in brain, this relatively minor pathway of APP metabolism within the ER/Golgi may have major implications for AD pathogenesis. All proteins destined for the cell membrane or secretion must first translocate into the ER. Newly translocated proteins are folded and assembled by a group of proteins, which include immunoglobulin-binding protein (BiP)/glucose-regulated...

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