Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70.

Abstract

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, andXenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes.However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.Numerous recent studies (2,7,8,10,34,49,54) have shown that one of the major heat shock proteins, hsp70, functions in an ATP-dependent manner through transient interactions to mediate folding or unfolding of polypeptide chains. Another major heat shock protein, hsp90, is thought to perhaps also function in some capacity related to folding or protein-protein interactions, but its function(s) remains poorly defined (1). Supporting its potential role in protein folding is a recent demonstration that hsp90 enhances renaturation of some proteins in vitro (52). Perhaps the most widely studied interaction of hsp90 is its identity as a stable component of several unactivated steroid receptor complexes (6, 38, 41). For glucocorticoid receptors, hsp90 binding to receptor is required to maintain high-affinity ligand binding (3,26,40), but other steroid receptors that have been examined do not show this same dependency on hsp90. In all hsp90-nuclear receptor complexes, ligand-dependent activation of the receptor DNA-binding ability is accompanied by dissociation of hsp90 (14,17,31,43), and it appears likely that one hsp90 function is to repress DNA binding by receptor.Steroid receptor-hsp9O interactions provide a model for understanding hsp90 function, but exploiting this model has been hindered by the inability to reversibly assemble receptor-hsp90 complexes in vitro. This drawback was recently overcome by establishing certain physicochemical conditions that permit the use of rabbit reticulocyte...

Firefly luciferase mutants as sensors of proteome stress

“…HOP, Hsp70-Hsp90 Organizing Protein, a 60 kDa protein (also called Sti1 or p60 in yeast) was first identified in a genetic screen to play a role in heat shock response of some Hsp70 genes (Nicolet and Craig, 1989;Smith et al, 1993). Subsequently, it was shown that HOP is a functional homolog of the BAG-1 protein that stimulates nucleotide exchange by Hsp70 (Gross and Hessefort, 1996).…”

1H, 15N and 13C backbone resonance assignments of the TPR1 and TPR2A domains of mouse STI1

“…Hop/STI1 (Hsp-organizing protein/stress-inducedphosphoprotein 1) is a key scaffold protein, which mediates client transfer from Hsp70 to Hsp90 in the later stages of client maturation (Smith et al 1993;Chen and Smith 1998). Initially, client proteins complex with Hsp40 and Hsp70 (Hernandez et al 2002;Pratt and Toft 2003).…”

Mammalian DNA cytosine‐5 methyltransferase interacts with p23 protein

“…p23, so named for its apparent molecular weight on SDS-PAGE gels, is an ~ 18 kDa protein of wide tissue distribution, which is highly conserved throughout eukaryotes, p23 was first observed as a component of the cytoplasmic form of the avian progesterone receptor (PR) complex [24], but it has also been found associated with the 90 kDa heat shock protein, hsp90 [25].…”