Heat Shock Protein 80 of <i>Neurospora crassa</i>, a Cytosolic Molecular Chaperone of the Eukaryotic Stress 90 Family, Interacts Directly with Heat Shock Protein 70

Freitag, Derrick G.; Ouimet, P M; Girvitz, T. L.; Kapoor, M.

doi:10.1021/bi963030g

Cited by 27 publications

(17 citation statements)

References 45 publications

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“…We have not observed any binding of hsp90 to hsp70 directly, in the absence of Hop. However, such an interaction has been suggested by chemical cross-linking studies (27) and has been observed directly with proteins from neurospora (64).…”

Section: Discussionmentioning

confidence: 99%

The Assembly and Intermolecular Properties of the hsp70-Hop-hsp90 Molecular Chaperone Complex

Hernandez

Sullivan

Toft

2002

Journal of Biological Chemistry

171

168

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The highly coordinated interactions of several molecular chaperones, including hsp70 and hsp90, are required for the folding and conformational regulation of a variety of proteins in eukaryotic cells, such as steroid hormone receptors and many other signal transduction regulators. The protein called Hop serves as an adaptor protein for hsp70 and hsp90 and is thought to optimize their functional cooperation. Here we characterize the assembly of the hsp70-Hop-hsp90 complex and reveal interactions that cause conformational changes between the proteins in the complex. We found that hsp40 plays an integral role in the assembly by enhancing the binding of hsp70 to the Hop complex. This is accomplished by stimulating the conversion of hsp70-ATP to hsp70-ADP, the hsp70 conformation favored for Hop binding. The hsp70-Hop-hsp90 complex is highly dynamic, as has been observed previously for hsp90 in its interaction with client proteins. Nonetheless, hsp90 binds with high affinity to Hop (K d ‫؍‬ 90 nM), and this binding is not affected by hsp70. hsp70 binds with lower affinity to Hop (K d ‫؍‬ 1.3 M) on its own, but this affinity is increased (K d ‫؍‬ 250 nM) in the presence of hsp90. hsp90 also reduces the number of hsp70 binding sites on the Hop dimer from two sites in the absence of hsp90 to one site in its presence. Hop can inhibit the ATP binding and p23 binding activity of hsp90, yet this can be reversed if hsp70 is present in the complex. Taken together, our results suggest that the assembly of hsp70-Hop-hsp90 complexes is selective and influences the conformational state of each protein.

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Section: Discussionmentioning

confidence: 99%

The Assembly and Intermolecular Properties of the hsp70-Hop-hsp90 Molecular Chaperone Complex

Hernandez

Sullivan

Toft

2002

Journal of Biological Chemistry

171

168

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“…In Neurospora, however, cytosolic Hsp90 and Hsp70 have been shown to interact directly (Freitag et al, 1997). To test whether HSP90C and HSP70B may also interact directly, we mixed heterologously expressed HSP90C (0.012 mM) and HSP70B (0.015 mM) in the presence and absence of Mg-ATP in vitro.…”

Section: Hsp90c Interacts With the Chloroplast Chaperone Hsp70bmentioning

confidence: 99%

HEAT SHOCK PROTEIN 90C Is a Bona Fide Hsp90 That Interacts with Plastidic HSP70B in Chlamydomonas reinhardtii

Willmund

Schroda

2005

Plant Physiology

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We report on the molecular and biochemical characterization of HEAT SHOCK PROTEIN 90C (HSP90C), one of the three Hsp90 chaperones encoded by the Chlamydomonas reinhardtii genome. Fractionation experiments indicate that HSP90C is a plastidic protein. In the chloroplast, HSP90C was localized to the soluble stroma fraction, but also to thylakoids and lowdensity membranes containing inner envelopes. HSP90C is expressed under basal conditions and is strongly induced by heat shock and moderately by light. In soluble cell extracts, HSP90C was mainly found to organize into dimers, but also into complexes of high molecular mass. Also, heterologously expressed HSP90C was mainly found in dimers, but tetramers and fewer monomers were detected, as well. HSP90C exhibits a weak ATPase activity with a K m for ATP of approximately 48 mM and a k cat of approximately 0.71 min 21 . This activity was inhibited by the Hsp90-specific inhibitor radicicol. In coimmunoprecipitation experiments, we found that HSP90C interacts with several proteins, among them plastidic HSP70B. The cellular concentration of HSP70B was found to be 2.9 times higher than that of HSP90C, giving a 4.8:1 stoichiometry of HSP70B monomers to HSP90C dimers. The strong inducibility of HSP90C by heat shock implies a role of the chaperone in stress management. Furthermore, its interaction with HSP70B suggests that, similar to their relatives in cytosol and the endoplasmic reticulum, both chaperones might constitute the core of a multichaperone complex involved in the maturation of specific client proteins, e.g. components of signal transduction pathways.

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“…hsp90 is a functional homodimer with one homodimerization domain in the C terminus (17) and a second located within the N terminus (16). There are also a number of co-chaperone binding domains, including one for the acidic phosphoprotein p23, which binds to the amino-terminal and central region of hsp90 in an ATP-dependent manner (13, 18 -21) and one for other co-chaperones (15,(22)(23)(24)(25).The best characterized domain of hsp90 is the highly conserved, N-terminal, nucleotide-binding pocket (12,13,26), which also serves as the binding site for the hsp90 inhibitor geldanamycin (GA) (13,27). GA, a benzoquinone ansamycin antibiotic, exerts its inhibitory effect by blocking the ATP-dependent binding of p23 to hsp90 (12, 13).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

The Biochemical Role of the Heat Shock Protein 90 Chaperone Complex in Establishing Human Telomerase Activity

Keppler¹,

A²,

Jarstfer

2006

Journal of Biological Chemistry

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Telomerase is a ribonucleoprotein complex that synthesizes the G-rich DNA found at the 3-ends of linear chromosomes. Human telomerase consists minimally of a catalytic protein (hTERT) and a template-containing RNA (hTR), although other proteins are involved in regulating telomerase activity in vivo. Several chaperone proteins, including hsp90 and p23, have demonstrable roles in establishing telomerase activity both in vitro and in vivo, and previous reports indicate that hsp90 and p23 are required for the reconstitution of telomerase activity from recombinant hTERT and hTR. Here we report that hTERT and hTR associate in the absence of a functional hsp90-p23 heterocomplex. We also report that hsp90 inhibitors geldanamycin and novobiocin inhibit recombinant telomerase even after telomerase is assembled. Inhibition by geldanamycin could be overcome by allowing telomerase to first bind its primer, suggesting a role for hsp90 in loading telomerase onto the telomere. Inhibition by novobiocin could not similarly be overcome but instead resulted in destabilization of the hTERT polypeptide. We propose that the hsp90-p23 complex fine tunes and stabilizes a functional telomerase structure, allowing primer loading and extension.Telomerase is an RNA-dependent DNA polymerase that extends the 3Ј-ends of linear chromosomes, allowing replicating cells to overcome the end replication problem (1). The complete subunit composition of the human telomerase ribonucleoprotein (RNP) 4 complex has yet to be fully elucidated, but the minimally active enzyme requires the reverse transcriptase catalytic subunit (hTERT) and the RNA subunit (hTR), which contains the template (2-5). Recent findings revealed that chaperone proteins, such as hsp90, p23, hsp70, p60, and hsp40/ydj, are also part of the telomerase RNP, at least during part of the cell cycle (6, 7). In fact, these chaperones are required to obtain a functionally active telomerase RNP in vitro, although their precise role in telomerase assembly remains enigmatic (6, 7). hsp90 is a highly conserved and abundant protein found in all eukaryotic cells (8, 9). hsp90 functions as part of a "foldosome" complex that together with other chaperones facilitates the accurate arrangement of numerous proteins (10, 11). This activity is dependent on a number of previously identified functional domains. Important for pharmacological concerns are the N-terminal ATP-binding site (12, 13) and a putative C-terminal ATP-binding site (14, 15). These sites are essential for hsp90 function, because ATP binding and hydrolysis are crucial in the conformational regulation of hsp90 and therefore its effects on client proteins (16). It has been suggested that the two ATP-binding sites act cooperatively, allowing cross-talk mediated by a central charged domain between the two termini (15). hsp90 is a functional homodimer with one homodimerization domain in the C terminus (17) and a second located within the N terminus (16). There are also a number of co-chaperone binding domains, including one for the acidic phos...

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Heat Shock Protein 80 of Neurospora crassa, a Cytosolic Molecular Chaperone of the Eukaryotic Stress 90 Family, Interacts Directly with Heat Shock Protein 70

Cited by 27 publications

References 45 publications

The Assembly and Intermolecular Properties of the hsp70-Hop-hsp90 Molecular Chaperone Complex

The Assembly and Intermolecular Properties of the hsp70-Hop-hsp90 Molecular Chaperone Complex

HEAT SHOCK PROTEIN 90C Is a Bona Fide Hsp90 That Interacts with Plastidic HSP70B in Chlamydomonas reinhardtii

The Biochemical Role of the Heat Shock Protein 90 Chaperone Complex in Establishing Human Telomerase Activity

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