In the eukaryotic cytosol, Hsp70 and Hsp90 cooperate with various co-chaperone proteins in the folding of a growing set of substrates, including the glucocorticoid receptor (GR). Here, we analyse the function of the co-chaperone Tpr2, which contains two chaperonebinding TPR domains and a DnaJ homologous J domain. In vivo, an increase or decrease in Tpr2 expression reduces GR activation, suggesting that Tpr2 is required at a narrowly de®ned expression level. As shown in vitro, Tpr2 recognizes both Hsp70 and Hsp90 through its TPR domains, and its J domain stimulates ATP hydrolysis and polypeptide binding by Hsp70. Furthermore, unlike other co-chaperones, Tpr2 induces ATP-independent dissociation of Hsp90 but not of Hsp70 from chaperone±substrate complexes. Excess Tpr2 inhibits the Hsp90-dependent folding of GR in cell lysates. We propose a novel mechanism in which Tpr2 mediates the retrograde transfer of substrates from Hsp90 onto Hsp70. At normal levels substoichiometric to Hsp90 and Hsp70, this activity optimizes the function of the multichaperone machinery. Keywords: glucocorticoid receptor/Hsp70/Hsp90/ molecular chaperones/protein folding Introduction Molecular chaperones aid in the folding of newlysynthesized polypeptides and the refolding of proteins after stress-induced denaturation. In the eukaryotic cytosol, the ATP-dependent chaperones Hsc70/Hsp70 (termed Hsp70 for simplicity) and Hsp90 often cooperate in the folding of a variety of substrate proteins. Hsp70 recognizes extended hydrophobic peptide sequences and generally acts at an early stage of polypeptide folding. In contrast, Hsp90 is thought to recognize the near-native conformations of a more restricted range of substrates, including several nuclear receptors (Bukau and Horwich, 1998;Buchner, 1999;Young et al., 2001; Hartl and HayerHartl, 2002).The function of the Hsp70/Hsp90 multichaperone machinery has been analysed in much detail for the progesterone receptor and glucocorticoid receptor (GR) (Pratt and Toft, 1997). After the initial binding of Hsp70, the co-chaperone Hop connects Hsp70 to Hsp90, which recognizes the ligand binding domain (LBD) of the receptor. Hsp70 and Hop are then replaced on the Hsp90±receptor complex by the immunophilin FKBP52 and the co-chaperone protein p23, and the receptor attains a state capable of binding the hormone ligand. In the presence of hormone, the receptor can proceed to activate transcription from speci®c steroid hormone response elements on the chromatin (Pratt and Toft, 1997;Chen and Smith, 1998;Johnson et al., 1998).For both Hsp70 and Hsp90, polypeptide binding and release is regulated by an ATPase cycle. Hsp70 in its ATPbound form has fast on-and off-rates for substrate polypeptides and ATP hydrolysis by Hsp70 is stimulated by the DnaJ homology domains (J domains) of Hsp40 and related co-chaperones. The ADP-bound state of Hsp70 binds substrates stably (Bukau and Horwich, 1998). Hsp90 also undergoes an ATP-regulated folding cycle, but unlike Hsp70, the substrate is held by the ATP-bound state of Hsp90 and ...

The von Hippel-Lindau tumor suppressor protein pVHL assembles with cullin-2 (hCUL-2) and elongin B͞C forming a protein complex, CBC VHL , that resembles SKP1-CDC53-F-box protein ubiquitin ligases. Here, we show that hCUL-2 is modified by the conserved ubiquitin-like protein NEDD8 and that NEDD8-hCUL-2 conjugates are part of CBC VHL complexes in vivo. Remarkably, the formation of these conjugates is stimulated by the pVHL tumor suppressor. A tumorigenic pVHL variant, however, is essentially deficient in this activity. Thus, ligation of NEDD8 to hCUL-2 is linked to pVHL activity and may be important for pVHL tumor suppressor function.

Heat shock protein 90 (HSP90) is considered a specialized molecular chaperone that controls the folding of cell-regulatory proteins such as steroid receptors and kinases. However, its high abundance is suggestive of a more general function in other fundamental processes. Here, we show that HSP90 is required for vesicular protein transport in the cell. We have identified a novel chaperone complex comprising HSP90 and TPR1 that is recruited to the membrane protein VAP-33. Depletion of the TPR1 protein in mammalian cells inhibits transport of vesicular stomatitis virus glycoprotein (VSVG) and leads to accumulation of this cargo protein in the Golgi apparatus. Furthermore, trafficking of VSVG between Golgi stacks is dependent on the ATPase function of HSP90 and can be inhibited by drugs specific for HSP90. Our results identify a new role for HSP90 in protein sorting, pointing to a central role for this molecular chaperone in the cell.

Mutations of the VHL tumor suppressor gene occur in patients with VHL disease and in the majority of sporadic clear cell renal carcinomas (VHL(-/-) RCC). Loss of VHL protein function is associated with constitutive expression of mRNAs encoding hypoxia-inducible proteins, such as vascular endothelial growth factor. Overproduction of angiogenic factors might explain why VHL(-/-) RCC tumors are so highly vascularized, but whether this overproduction is sufficient for oncogenesis still remains unknown. In this report, we examined the activity of transforming growth factor-alpha (TGF-alpha), another VHL-regulated growth factor. We show that TGF-alpha mRNA and protein are hypoxia-inducible in VHL(-/-) RCC cells expressing reintroduced VHL. In addition to its overexpression by VHL(-/-) RCC cells, TGF-alpha can also act as a specific growth-stimulatory factor for VHL(-/-) RCC cells expressing reintroduced wild-type VHL, as well as primary renal proximal tubule epithelial cells, the likely site of origin of RCC. This role is in contrast to those of other growth factors overexpressed by VHL(-/-) RCC cells, such as vascular endothelial growth factor and TGF-beta1, which do not stimulate RCC cell proliferation. A TGF-alpha-specific antisense oligodeoxynucleotide blocked TGF-alpha production in VHL(-/-) RCC cells, which led to the dependence of those cells on exogenous growth factors to sustain growth in culture. Growth of VHL(-/-) RCC cells was also significantly reduced by a drug that specifically inhibits the epidermal growth factor receptor, the receptor through which TGF-alpha stimulates proliferation. These results suggest that the generation of a TGF-alpha autocrine loop as a consequence of VHL inactivation in renal proximal tubule epithelial cells may provide the uncontrolled growth stimulus necessary for the initiation of tumorigenesis.