Complexes between nascent polypeptides and their molecular chaperones in the cytosol of mammalian cells.

Abstract

Folding of newly synthesized proteins in vivo is believed to be facilitated by the cooperative interaction of a defined group of proteins known as molecular chaperones.We investigated the direct interaction of chaperones with nascent polypeptides in the cytosol of mammalian cells by multiple methods. A new approach using a polyclonal antibody to puromycin allowed us to tag and capture a population of truncated nascent polypeptides with no bias as to the identity of the bound chaperones. In addition, antibodies that recognize the cytosolic chaperones hsp7O, CCT (TRiC), hsp4O, p48 (Hip), and hsp9O were compared on the basis of their ability to coprecipitate nascent polypeptides, both before and after chemical cross-linking. By all three approaches, hsp7O was found to be the predominant chaperone bound to nascent polypeptides. The interaction between hsp7O and nascent polypeptides is apparently dynamic under physiological conditions but can be stabilized by depletion of ATP or by cross-linking. The cytosolic chaperonin CCT was found to bind primarily to full-length, newly synthesized actin, and tubulin. We demonstrate and caution that nascent polypeptides have a propensity for binding many proteins nonspecifically in cell lysates. Although current models of protein folding in vivo have described additional components in contact with nascent polypeptides, our data indicate that the hsp7O and, perhaps, the hsp9O families are the predominant classes of molecular chaperones that interact with the general population of cytosolic nascent polypeptides.

Evidence that Hsc70 negatively modulates the activation of the heme‐regulated eIF‐2α kinase in rabbit reticulocyte lysate

“…SDS/PAGE of approximately 10 µg of the Hsc70 preparation indicated that the Hsc70 was highly pure (Ͼ 99%), as judged by the absence of any other Coommassie blue staining bands. Western blot analysis of the Hsc70 preparation using a polyclonal anti-DnaJ serum [26] did not detect the presence of any contaminating DnaJ homologs.…”

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

“…Factors associated to newly synthesized polypeptides are typical examples. Newly synthesized polypeptides emerging form the ribosomes can be immobilized on protein complexes associated with ribosomes using chemical cross-linkers such as 3,3´-dithiobis[sulfosuccinimidylpropionate] (DTSSP) 31 . This approach revealed that the Receptor for Activated C Kinase 1 (RACK1)/c-Jun N-terminal kinase (JNK)/eukaryotic translation elongation factor 1A2 (eEF1A2) complex regulates degradation of newly synthesized polypeptides in response to stress 27 .…”

Fine-Tuning of Translation Termination Efficiency inSaccharomyces cerevisiaeInvolves Two Factors in Close Proximity to the Exit Tunnel of the Ribosome

“…This suggests that Ssbp functions as a chaperone for polypeptide chains during translation. Such a role in emerging polypeptides could be to facilitate the successful folding of newly synthesized proteins (Beckmann et al 1990;Frydman et al 1994;Hardesty et al 1995;Eggers et al 1997). An alternative explanation of the role of the Ssb chaperone would be through a direct role in the decoding process.…”