Effect of yeast and human DnaJ homologs on clathrin uncoating by 70 kilodalton heat shock protein

We recently found that the DnaJ homolog auxilin is required for Hsc70 to uncoat clathrin baskets. In the present study, we investigated the effect of two other DnaJ homologs, YDJ1 from yeast and HDJ1 from humans, on the uncoating activity of Hsc70. Neither YDJ1 nor HDJ1 substituted for auxilin in supporting uncoating. Rather, in the presence of auxilin, both YDJ1 and HDJ1 strongly inhibited uncoating at pH 7 and also prevented the binding of Hsc70 to clathrin baskets at pH 6. Both YDJ1, as shown previously, and HDJ1 catalytically induce polymerization of Hsc70 into large polymers in ATP, and the YDJ1 concentration required to inhibit uncoating was similar to the concentration required for polymerization. However, uncoating was almost completely inhibited even at low concentrations of Hsc70 where only partial polymerization occurs, suggesting that YDJ1 inhibits uncoating not only by polymerizing the Hsc70 but also by some other mechanism as well. The effects of YDJ1 and HDJ1 were completely reversible; when they were removed, the Hsc70 regained full activity. Since both YDJ1 and HDJ1 inhibited the uncoating of clathrin baskets by brain cytosol as well as by purified Hsc70, this could be a physiological phenomenon which could affect other activities of Hsc70 in addition to uncoating.     

Characterization of D10S and K71E mutants of human cytosolic hsp70

To determine the effect of mutations at the nucleotide-binding site of recombinant Hsp70 on its interaction with protein and peptide substrates, point mutations were made at D10 and K71, two residues at the active site. The D10S mutation weakened both ATP and ADP binding, while the K71E mutation weakened only ATP binding. In binding experiments using Hsp70 with no bound nucleotide, the mutated Hsp70s interacted with clathrin and peptide just like the wild-type Hsp70. However, the D10 mutation completely abolished the effects of both ATP and ADP on peptide and clathrin binding. The K71 mutation also abolished the effect of ATP on substrate binding, but ADP, which still bound tightly, had its normal effect on substrate binding. In addition, the D10S and K71E mutants had greatly reduced ability to uncoat clathrin-coated vesicles at pH 7.0, bind to clathrin baskets at pH 6.0, and undergo polymerization induced by YDJ1 in the presence of ATP. We conclude, first, that nucleotides must bind strongly to Hsp70 to affect substrate binding and, second, that interaction of Hsp70 with DnaJ homologues may also require a strongly bound ATP.     

Purification of a 38-kDa protein from rabbit reticulocyte lysate which promotes protein renaturation by heat shock protein 70 and its identification as delta-aminolevulinic acid dehydratase and as a putative DnaJ protein

We reported recently that a rabbit reticulocyte 66-kDa protein (termed RF-hsp 70 by our laboratory and p60 and hop by others) functions as a hsp 70 recycling protein and markedly enhances the renaturation of luciferase by hsp 70 (Gross, M., and Hessefort, S. (1996) J. Biol. Chem. 271, 16833-16841). In this report, we confirm that the ability of RF-hsp 70 to promote the conversion of hsp 70. ADP to hsp 70.ATP, thus enhancing the protein folding activity of hsp 70, is caused by the purified 66-kDa protein and not by a trace DnaJ/hsp 40 protein contaminant. To determine the relationship between RF-hsp 70 and the DnaJ/hsp 40 heat shock protein family, which also enhances protein renaturation by hsp 70, we purified a 38-kDa protein from rabbit reticulocyte lysate based upon its ability to stimulate renaturation of luciferase by hsp 70. Partial amino acid sequencing of this 38-kDa protein has indicated, unexpectedly, that it is the enzyme delta-aminolevulinic acid dehydratase (ALA-D) and that it does not contain detectable sequences corresponding to the DnaJ/hsp 40 protein family. In addition, immunoblot analysis with a polyclonal antibody made to HeLa cell hsp 40 (from StressGen) confirms that our purified ALA-D contains no hsp 40, although hsp 40 is present in relatively crude rabbit reticulocyte protein fractions. Rabbit reticulocyte ALA-D is about as active in converting delta-aminolevulinic acid to porphobilinogen and as Zn2+-dependent as ALA-D purified from other sources. Rabbit reticulocyte ALA-D stimulates the renaturation of luciferase by hsp 70 up to 10-fold at concentrations that are the same as or less than that of hsp 70, and it has no renaturation activity in the absence of hsp 70. The renaturation effect of ALA-D is additive with that of RF-hsp 70 at limiting or saturating concentrations of each, and, unlike RF-hsp 70, ALA-D does not promote the dissociation of hsp 70.ADP in the presence of ATP. The renaturation-enhancing effect of ALA-D may be caused by a region near its carboxyl terminus which has sequence homology to the highly conserved domain of the DnaJ protein family, which is similar to the sequence homology between this domain and a carboxyl-terminal region in auxilin, a DnaJ-like protein that requires this region for its hsp 70-dependent function (Ungewickell, E., Ungewickell, H., Holstein, S. E. H., Lindner, R., Prasad, K., Barouch, W., Martin, B., Greene, L. E., and Eisenberg, E. (1995) Nature 378, 632-635).     

Clathrin assembly protein AP180: primary structure, domain organization and identification of a clathrin binding site

Binding of AP180 to clathrin triskelia induces their assembly into 60-70 nm coats. The largest rat brain cDNA clone isolated predicts a molecular weight of 91,430 for AP180. Two cDNA clones have an additional small 57 bp insert. The deduced molecular weight agrees with gel filtration results provided the more chaotropic denaturant 6 M guanidinium thiocyanate is substituted for the weaker guanidinium chloride. The sequence and the proteolytic cleavage pattern suggest a three domain structure. The N-terminal 300 residues (pI 8.7) harbour a clathrin binding site. An acidic middle domain (pI 3.6, 450 residues), interrupted by an uncharged alanine rich segment of 59 residues, appears to be responsible for the anomalous physical properties of AP180. The C-terminal domain (166 residues) has a pI of 10.4. AP180 mRNA is restricted to neuronal sources. AP180 shows no significant homology to known clathrin binding proteins, but is nearly identical to a mouse phosphoprotein (F1-20). This protein, localized to synaptic termini, has so far been of unknown function.     

Arrestin/clathrin interaction. Localization of the arrestin binding locus to the clathrin terminal domain

Previously we demonstrated that nonvisual arrestins exhibit a high affinity interaction with clathrin, consistent with an adaptor function in the internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this report we show that a short sequence of highly conserved residues within the globular clathrin terminal domain is responsible for arrestin binding. Limited proteolysis of clathrin cages results in the release of terminal domains and concomitant abrogation of arrestin binding. The nonvisual arrestins, beta-arrestin and arrestin3, but not visual arrestin, bind specifically to a glutathione S-transferase-clathrin terminal domain fusion protein. Deletion analysis and alanine scanning mutagenesis localize the binding site to residues 89-100 of the clathrin heavy chain and indicate that residues 1-100 can function as an independent arrestin binding domain. Site-directed mutagenesis identifies an invariant glutamine (Glu-89) and two highly conserved lysines (Lys-96 and Lys-98) as residues critical for arrestin binding, complementing hydrophobic and acidic residues in arrestin3 which have been implicated in clathrin binding (Krupnick, J. G., Goodman, O. B., Jr., Keen, J. H., and Benovic, J. L. (1997) J. Biol. Chem. 272, 15011-15016). Despite exhibiting high affinity clathrin binding, arrestins do not induce coat assembly. The terminal domain is oriented toward the plasma membrane in coated pits, and its binding of both arrestins and AP-2 suggests that this domain is the anchor responsible for adaptor-receptor recruitment to the coated pit.     

Binding of heptapeptides or unfolded proteins to the chimeric C-terminal domains of 70-kDa heat shock cognate protein

Seventy-kDa heat shock cognate protein (hsc70) and its homologs in bacteria, yeast and vertebrates are known to form complexes with S-carboxymethyl-alpha-lactalbumin (CMLA), an unfolded protein; and, this activity has been attributed to its C-terminal 30-kDa domain. Herein, we show that hsc70s isolated from the seeds of mung bean and peas, however, are not effective in complexing with CMLA, and that the 30-kDa domain of Arabidopsis hsc70 (At30) cannot form stable complexes with CMLA either. Moreover, chimeric 30-kDa domains, either composed of rat 18-kDa and Arabidopsis 10-kDa subdomains (R18At10) or with Arabidopsis 18-kDa and rat 10-kDa subdomains (At18R10), were prepared and tested for their ability to complex with CMLA or a heptapeptide FYQLALT. At18R10 cannot complex with both CMLA and FYQLALT. On the other hand, R18At10 is capable of forming complexes with FYQLALT at a level similar to that of the rat 30-kDa domain (R30). R18At10 also forms complexes with CMLA, but the amount of the R18At10/CMLA complexes is much less than that of R30/CMLA. The results imply that the 18-kDa subdomain dictates the binding specificity for heptapeptide, and that the C-terminal 10-kDa subdomain may also provide some selection or restriction for unfolded proteins to form complexes with hsc70.     

ATP- and cytosol-dependent release of adaptor proteins from clathrin-coated vesicles: A dual role for Hsc70

Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP- and cytosol- dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release.     

Clathrin-coated pit-associated proteins are required for alveolar macrophage phagocytosis

During phagocytosis, phagocytic receptors and membrane material must be inserted in the pseudopod membrane as it extends over the phagocytic target. This may require a clathrin-mediated recycling mechanism similar to that postulated for leading edge formation during cell migration. To investigate this possibility, liposomes were used to deliver to intact rat alveolar macrophages (AMs): 1) Abs to clathrin, clathrin adaptor AP-2, and hsc70, and 2) amantadine. Phagocytosis was assayed by fluorometric and colorimetric techniques. Liposome-delivered Abs to clathrin and AP-2 inhibited AM phagocytosis of zymosan-coated, fluorescent liposomes from 16.3+/-0.3 to 5.8+/-0.3, and 10.1+/-0.9 to 4.8+/-0.2 liposomes/cell (p<0.01). Similarly, liposome-delivered Ab to clathrin also inhibited AM phagocytosis of IgG-opsonized RBCs from 11.7+/-1.7 to 3.8+/-0.7 RBCs/cell (p<0.01). Amantadine, which blocks the budding of clathrin-coated vesicles, inhibited phagocytosis from 13.8+/-0.8 to 5.7+/-0.6 (p<0.01). Ab blockade of hsc70, which catalyzes clathrin turnover, also inhibited phagocytosis from 9.1+/-0.5 to 4.3+/-0.2 (p<0.01). These findings suggest that clathrin-mediated receptor/membrane recycling is required for phagocytosis.     

J proteins catalytically activate Hsp70 molecules to trap a wide range of peptide sequences

Proteins of the Hsp70 family of ATPases, such as BiP, function together with J proteins to bind polypeptides in numerous cellular processes. Using a solid phase binding assay, we demonstrate that a conserved segment of the J proteins, the J domain, catalytically activates BiP molecules to bind peptides in its immediate vicinity. The J domain interacts with the ATP form of BiP and stimulates hydrolysis resulting in the rapid trapping of peptides, which are then only slowly released upon nucleotide exchange. Activation by the J domain allows BiP to trap peptides or proteins that it would not bind on its own. These results explain why BiP and probably all other Hsp70s can interact with a wide range of substrates and suggest that the J partner primarily determines the substrate specificity of Hsp70s.     

Aggregation of hsp70 and hsc70 in vivo is distinct and temperature-dependent and their chaperone function is directly related to non-aggregated forms

We used non-denaturing gradient analysis of cell extracts before and after heat treatment of the cells and showed that hsp70 and hsc70 aggregate in vivo in a temperature-dependent fashion. Their aggregation profiles were found to be clearly distinguishable and sensitive to ATP depletion. Pore exclusion limit electrophoresis showed that these two proteins are mainly found in autoaggregated forms including dimers, trimers and oligomers. The addition of denatured luciferase to the cell extracts reversed the aggregation of both proteins towards their non-aggregated forms. Immunoprecipitation and Western-blot analysis showed that the non-aggregated form is the only one bound to denatured luciferase. Our results suggest that aggregated hsp70 and hsc70 represent predominantly self-associated molecules unable to exert chaperone activity. The cochaperone hsp40 was also found to be aggregated and, on addition of denatured luciferase, its aggregation was reversed to a non-aggregated state. Immunoprecipitation analysis indicated that hsp40 forms a complex with the non-aggregated form of hsc70 and denatured luciferase. These results confirm previous in vitro studies and support the suggestion that in vivo cytosolic hsp70 and hsc70 exist mainly in an oligomer-monomer equilibrium which is dependent on the environmental temperature, the levels of ATP and the presence of denatured proteins.     

ATP-binding site of human brain hexokinase as studied by molecular modeling and site-directed mutagenesis

The interaction of ATP with the active site of hexokinase is unknown since the crystal structure of the hexokinase-ATP complex is unavailable. It was found that the ATP binding site of brain hexokinase is homologous to that of actin, heat shock protein hsc70, and glycerol kinase. On the basis of these similarities, the ATP molecule was positioned in the catalytic domain of human brain hexokinase, which was modeled from the X-ray structure of yeast hexokinase. Site-directed mutagenesis was performed to test the function of residues presumably involved in interaction with the tripolyphosphoryl moiety of ATP. Asp532, which is though to be involved in binding the Mg2+ ion of the MgATP2- complex, was mutated to Lys and Glu. The kcat values decreased 1000- and 200-fold, respectively, for the two mutants. Another residue, Thr680 was proposed to interact with the gamma-phosphoryl group of ATP through hydrogen bonds and was mutated to Val and Ser. The kcat value of the Thr680Val mutant decreased 2000-fold, whereas the kcat value of the Thr680Ser decreased only 2.5-fold, implying the importance of the hydroxyl group. The Km and dissociation constant values for either ATP or glucose of all the above mutants showed little or no change relative to the wild-type enzyme. The Ki values for the glucose 6-phosphate analogue 1,5-anhydroglucitol 6-phosphate, were the same as that of the wild-type enzyme, and the inhibition was reversed by inorganic phosphate (Pi) for all four mutants. The circular dichroism spectra of the mutants were the same as that of the wild-type enzyme. The results from the site-directed mutagenesis demonstrate that the presumed interactions of investigated residues with ATP are important for the stabilization of the transition state.     

Platelet adhesion to collagen activates a phosphoprotein complex of heat-shock proteins and protein phosphatase 1

Rapid activation of blood platelets is required for effective haemostasis, with shape change, aggregation, secretion of granule contents and cell adhesion occurring in seconds or even milliseconds. Signal-transduction events, evidenced by changes in protein phosphorylation and calcium levels, also take place in this time domain. We have now shown that platelet adhesion to collagen via the alpha 2 beta 1 integrin under arterial shear forces initiated the rapid dephosphorylation of a 67 kDa protein "band" which contained the 70 kDa constitutive heat-shock protein, hsc70. Immunoprecipitation with hsc70 antibodies revealed a large phosphoprotein complex in resting platelets and adhesion caused dissociation of the complex along with dephosphorylation of hsc70. The complex also contained the hsp90 heat-shock protein, protein phosphatase IC, alpha, delta and M subunits, and some 7-8 unidentified phosphoproteins. The data suggest that heat-shock proteins and protein phosphatases are actively involved in integrin-mediated platelet adhesion.     

Isolation and characterization of a DnaJ-like protein in rats: the C-terminal 10-kDa domain of hsc70 is not essential for stimulating the ATP-hydrolytic activity of hsc70 by a DnaJ-like protein

A DnaJ-like protein, RDJ1, was isolated from a rat brain cDNA library. The protein is predicted to have 397 amino acid residues and shares 99% identity to that of HDJ2, a human DnaJ-like protein. RDJ1 was also shown to rescue the temperature-sensitive lethality of a strain containing a mutated cytosolic DnaJ in yeast, ydj1-151. Fragments containing the J-domain of RDJ1 either with or without the G/F motif were expressed in Escherichia coli. The purified proteins stimulated the ATPase activity of hsc70 and of the 60-kDa N-terminal fragment of hsc70. These results imply that RDJ1 can interact with the N-terminal 60-kDa fragment of hsc70 to activate ATP hydrolysis by hsc70.     

Destabilization of peptide binding and interdomain communication by an E543K mutation in the bovine 70-kDa heat shock cognate protein, a molecular chaperone

We have compared 70-kDa heat shock cognate protein (Hsc70) isolated from bovine brain with recombinant wild type protein and mutant E543K protein (previously studied as wild type in our laboratory). Wild type bovine and recombinant protein differ by posttranslational modification of lysine 561 but interact similarly with a short peptide (fluorescein-labeled FYQLALT) and with denatured staphylococcal nuclease-(Delta135-149). Mutation E543K results in 4. 5-fold faster release of peptide and lower stability of complexes with staphylococcal nuclease-(Delta135-149). ATP hydrolysis rates of the wild type proteins are enhanced 6-10-fold by the addition of peptide. The E543K mutant has a peptide-stimulated hydrolytic rate similar to that of wild type protein but a higher unstimulated rate, yielding a mere 2-fold enhancement. All three versions of Hsc70 possess similar ATP-dependent conformational shifts, and all show potassium ion dependence. These data support the following model: (i) in the presence of K+, Mg2+, and ATP, the peptide binding domain inhibits the ATPase; (ii) binding of peptide relieves this inhibition; and (iii) the E543K mutation significantly attenuates the inhibition by the peptide binding domain and destabilizes Hsc70-peptide complexes.     

Molybdenum cofactor biosynthesis. The plant protein Cnx1 binds molybdopterin with high affinity

The molybdenum cofactor is an essential part of all eukaryotic molybdoenzymes. It is a molybdopterin (MPT) revealing the same core structure in all organisms. The plant protein Cnx1 from Arabidopsis thaliana is involved in the multi-step biosynthesis of molybdenum cofactor. Previous studies (Stallmeyer, B., Nerlich, A., Schiemann, J., Brinkmann, H., and Mendel, R. R. (1995) Plant J. 8, 751-762) suggested a function of Cnx1 in a late step of cofactor biosynthesis distal to the formation of MPT, i.e. conversion of MPT into molybdenum cofactor. Here we present the first biochemical evidences confirming this assumption. The protein Cnx1 consists of two domains (E and G) homologous to two distinct Escherichia coli proteins involved in cofactor synthesis. Binding studies with recombinantly expressed and purified Cnx1 and with its single domains revealed a high affinity of the G domain to MPT (kD = 0.1 microM) with equimolar binding. In contrast, the E domain of Cnx1 binds MPT with lower affinity (kD = 1.6 microM) and in a cooperative manner (nH = 1. 5). The entire Cnx1 showed a tight and cooperative MPT binding. Based on these data providing a common link between both domains that matches the previous characterization of plant and bacterial Cnx1 homologous mutants, we present a model for the function of Cnx1.     

Characterization of dominant negative arrestins that inhibit beta2-adrenergic receptor internalization by distinct mechanisms

Arrestins have been shown to act as adaptor proteins that mediate the interaction of G protein-coupled receptors with the endocytic machinery. In this study, the role of arrestin-3 in receptor internalization was investigated by constructing different arrestin-3 minigenes that could potentially act as dominant negative inhibitors of arrestin function. Expression of arrestin-3 proteins containing amino acids 1-320 or 201-409 resulted in the inhibition of beta2-adrenergic receptor internalization in HEK-293 cells by approximately 40%. Both of these arrestins were diffusely localized within the cytoplasm of transfected cells, were unable to mediate redistribution of receptors to clathrin-coated pits, and did not localize to coated pits in either the presence or absence of receptor and agonist. Arrestin-3(1-320), but not arrestin-3(201-409), bound to light-activated phosphorylated rhodopsin with an affinity comparable with that of wild-type arrestin-3. In contrast, expression of arrestin-3 proteins composed of only the clathrin binding domain, arrestin-3(284-409), and arrestin-3(290-409) resulted in the constitutive localization of these arrestins to coated pits. Arrestin-3(284-409) and arrestin-3(290-409) acted as dominant negative inhibitors of wild-type arrestin function, inhibiting receptor internalization by 70 and 30%, respectively. Carboxyl-terminal deletions of arrestin-3 retained the ability to promote internalization until residues amino-terminal to amino acid 350 were deleted, suggesting that residues in this region also compose part of the clathrin binding domain in addition to the major binding site between residues 371-379. These studies characterize at least two distinct mechanisms, competition for either receptor or clathrin binding, by which dominant negative arrestins inhibit receptor internalization and further define residues within arrestin-3 that constitute the clathrin binding domain.     

REM sleep behavior disorder and degenerative dementia: an association likely reflecting Lewy body disease

It is commonly believed that MgATP2- is the substrate of F1-ATPases and ATP4- acts as a competitive inhibitor. However, the velocity equation for such competitive inhibition is equivalent to that for a rapid equilibrium ordered binding mechanism in which ATP4- adds first and the binding of Mg2+ is dependent on the formation of the E x ATP4- complex. According to this ordered-binding model, solution formed MgATP2- is not recognized by the ATPase as a direct substrate, and the high-affinity binding of Mg2+ to the E x ATP4- complex is the key reaction towards the formation of the ternary complex. These models (and others) were tested with an F1- ATPase, isolated from Halobacterium saccharovorum, by evaluating the rate of ATP hydrolysis as a function of free [ATP4-] or free [Mg2+]. The rates were asymmetrical with respect to increasing [ATP4-] versus increasing [Mg2+]. For the ordered-binding alternative, a series of apparent dissociation constants were obtained for ATP4-(K(A)aPP), which decreased as [Mg2+] increased. From this family of K(A)aPP the true K(A) was retrieved by extrapolation to [Mg2+] = 0 and was found to be 0.2 mM. The dissociation constants for Mg2+, established from these experiments, were also apparent (K(B)aPP) and dependent on [ATP4-] as well as on the pH. The actual K(B) was established from a series of K(B)aPP by extrapolating to [ATP4-] = infinity and to the absence of competing protons, and was found to be 0.0041 mM. The pKa of the protonable group for Mg2+ binding is 8.2. For the competitive inhibition alternative, rearrangement of the constants and fitting to the velocity equation gave an actual binding constant for MgATP2- (K(EAB)) of 0.0016 mM and for ATP4- (K(EA)) of 0.2 mM. Decision between the two models has far-reaching mechanistic implications. In the competitive inhibition model MgATP2- binds with high affinity, but Mg2+ cannot bind once the E x ATP4- complex is formed, while in the ordered-binding model binding of Mg2+ requires that ATP4- adds first. The steric constraints evident in the diffraction structure of the ATP binding site in the bovine mitochondrial F-ATPase [Abrahams, J. P., Leslie, A. G. W., Lutter, R. & Walker, J. E. (1994) Nature 370, 621-628] tend to favor the ordered-binding model, but the final decision as to which kinetic model is valid has to be from further structural studies. If the ordered-binding model gains more experimental support, a revision of the current concepts of unisite catalysis and negative cooperativity of nucleotide binding will be necessary.     

The hydroxyl of threonine 13 of the bovine 70-kDa heat shock cognate protein is essential for transducing the ATP-induced conformational change

The mechanism by which ATP binding transduces a conformational change in 70-kDa heat shock proteins that results in release of bound peptides remains obscure. Wei and Hendershot demonstrated that mutating Thr37 of hamster BiP to glycine impeded the ATP-induced conformational change, as monitored by proteolysis [(1995) J. Biol. Chem. 270, 26670-26676]. We have mutated the equivalent resitude of the bovine heat shock cognate protein (Hsc70), Thr13, to serine, valine, and glycine. Solution small-angle X-ray scattering experiments on a 60-kDa fragment of Hsc70 show that ATP binding induces a conformational change in the T13S mutant but not the T13V or T13G mutants. The kinetics of ATP-induced tryptophan fluorescence intensity changes in the 60-kDa proteins is biphasic for the T13S mutant but monophasic for T13V or T13G, consistent with a conformational change following initial ATP binding in the T13S mutant but not the other two. Crystallographic structures of the ATPase fragments of the T13S and T13G mutants at 1.7 A resolution show that the mutations do not disrupt the ATP binding site and that the serine hydroxyl mimics the threonine hydroxyl in the wild-type structure. We conclude that the hydroxyl of Thr13 is essential for coupling ATP binding to a conformational change in Hsc70. Molecular modeling suggests this may result from the threonine hydroxyl hydrogen-bonding to a gamma-phosphate oxygen of ATP, thereby inducing a structural shift within the ATPase domain that couples to its interactions with the peptide binding domain.     

Inhibition of phospholipase D by clathrin assembly protein 3 (AP3)

In the accompanying paper (Chung, J.-K., Sekiya, F., Kang, H.-S., Lee, C., Han, J.-S., Kim, S. R., Bae, Y. S., Morris, A. J., and Rhee, S. G. (1997) J. Biol. Chem. 272, 15980-15985), synaptojanin is identified as a protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2). Here, the purification from rat brain cytosol of another PLD-inhibitory protein that is immunologically distinct from synaptojanin is described, and this protein is identified as clathrin assembly protein 3 (AP3) by peptide sequencing and immunoblot analysis. AP3 binds both inositol hexakisphosphate and preassembled clathrin cages with high affinity. However, neither inositol hexakisphosphate binding nor clathrin cage binding affected the ability of AP3 to inhibit PLD. AP3 also binds to PI(4,5)P2 with low affinity. But the PI(4,5)P2 binding was not responsible for PLD inhibition, because the potency and efficacy of AP3 as an inhibitor of PLD were similar in the absence and presence of PI(4,5)P2. A bacterially expressed fusion protein, glutathione S-transferase-AP3 (GST-AP3), also inhibited PLD with a potency equal to that of brain AP3. The inhibitory effect of AP3 appeared to be the result of direct interaction between AP3 and PLD because PLD bound GST-AP3 in an in vitro binding assay. Using GST fusion proteins containing various AP3 sequences, we found that the sequence extending from residues Pro-290 to Lys-320 of AP3 is critical for both inhibition of and binding to PLD. The fact that AP3 is a synapse-specific protein indicates that the AP3-dependent inhibition of PLD might play a regulatory role that is restricted to the rapid cycling of synaptic vesicles.     

The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system DnaK, DnaJ, and GrpE

Molecular chaperones of the Hsp70 class bind unfolded polypeptide chains and are thought to be involved in the cellular folding pathway of many proteins. DnaK, the Hsp70 protein of Escherichia coli, is regulated by the chaperone protein DnaJ and the cofactor GrpE. To gain a biologically relevant understanding of the mechanism of Hsp70 action, we have analyzed a model reaction in which DnaK, DnaJ, and GrpE mediate the folding of denatured firefly luciferase. The binding and release of substrate protein for folding involves the following ATP hydrolysis-dependent cycle: (i) unfolded luciferase binds initially to DnaJ; (ii) upon interaction with luciferase-DnaJ, DnaK hydrolyzes its bound ATP, resulting in the formation of a stable luciferase-DnaK-DnaJ complex; (iii) GrpE releases ADP from DnaK; and (iv) ATP binding to DnaK triggers the release of substrate protein, thus completing the reaction cycle. A single cycle of binding and release leads to folding of only a fraction of luciferase molecules. Several rounds of ATP-dependent interaction with DnaK and DnaJ are required for fully efficient folding.