Principles of chaperone-assisted protein folding: Differences between in vitro and in vivo mechanisms

Science

Volumn 272, Issue 5267, 1996, Pages 1497-1502

  Frydman, Judith ^a,b   Hartl, F Ulrich ^a  

^a HOWARD HUGHES MEDICAL INSTITUTE   (United States)

^b STANFORD UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CHAPERONE; HEAT SHOCK PROTEIN; OLIGOMER; PROTEIN SUBUNIT;

ARTICLE; CYTOSOL; EUKARYOTIC CELL; PRIORITY JOURNAL; PROTEIN DENATURATION; PROTEIN FOLDING;

EID: 0029980091     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5267.1497     Document Type: Article

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29. 35S-labeled actin as a control. Identical results were obtained with either actin folding assay. Firefly luciferase was purchased from Sigma. Luciferase folding was assayed as described with the use of a commercially available assay system (Promega) as in (8).
30. 2, 0.5 mM EDTA, and 1 mM DTT). After removing protein aggregates by centrifugation (20,000g for 15 min at 4°C), refolding was initiated by addition of 1 mM ATP and an ATP regenerating system [creatine kinase (50 μg/ml) and 8 mM phosphocreatine], as described [E. Nimmesgern and F. U. Hartl, FEBS Lett. 331, 25 (1993)]. At different time points, the reactions were stopped on ice by the addition of 10 mM trans-1,2-cyclohexanediaminetetraacetate (CDTA). Both luciferase and actin aggregated when diluted into either buffer A or buffer A containing bovine serum albumin (5 mg/ml). Coimmunoprecipitation with chaperone antibodies was performed as described after stopping the reactions with apyrase (0.5 U/μl) (8).
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34. The addition of T-GroEL at 1 and 3 μM reduced the efficiency of translation by ∼30%, which explains the decrease in the amount of native actin seen in Fig. 3D. Quantification of the amount of full-length actin in these translations indicated that there was no decrease in the fraction of folded actin (see Fig. 3A). Addition of T-GroEL reduced the amount of TRiC-bound actin chains because the translation reaction contains incomplete chains that are unable to fold. In contrast to T-GroEL, addition of T-TRiC [see (25) and Fig. 4B] inhibited the folding of actin when added from the beginning of translation.
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38. Hemin is essential for translation in reticulocytes but inhibits proteasome function [A. L. Haas and I. A. Rose, Proc. Natl. Acad. Sci. U.S.A. 78, 6845 (1981)]; we removed it by desalting the lysates containing ribosome-associated actin-ΔC on Sephadex G-25 columns equilibrated in buffer A. Actin-ΔC was degraded through the proteasome-ubiquitin pathway, because degradation was inhibited by hemin (with the accumulation of ubiquitin conjugates) and by methylated ubiquitin [A. Hershko, D. Ganoth, J. Pehrson, R. E. Palazzo, L. H. Cohen, J. Biol. Chem. 266, 16376 (1991)]. Similar results were obtained for actin-ΔC 1-220 and actin-ΔC 1-368.
39. The protection of nascent actin chains against degradation was underestimated because of the spontaneous release of nascent chains from the ribosome at 37°C (Fig. 3E), which we measured by including hemin during the incubation with cycloheximide and separating ribosome-bound and released chains by sedimentation through a dense sucrose solution (19).
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41. Evidence has been presented that during TRiC-mediated folding, actin undergoes cycles of ATP-dependent release and rebinding involving its discharge into the solution in a non-native state [G. Tian, I. E. Vainberg, W. D. Tap, S. A. Lewis, N. J. Cowan, Nature 375, 250 (1995)].
42. 2, 10% glycerol, and 1 mM DTT]. T-TRiC was prepared by incubating TRiC in buffer D with 2 mM 8-azido-ATP in the dark for 5 min at 30°C, followed by crosslinking [by exposure to UV light (310 to 340 nm) for 10 min at 4°C] and repurification. Folding reactions were started by addition of 1 mM ATP and were incubated for 40 min at 30°C.
43. We confirmed the previous observation that actin can be released from TRiC in a non-native state (24) with a diluted actin-TRiC complex (25 nM) under conditions where we found bovine TRiC to be structurally labile. The lack of inhibition of actin folding by T-GroEL observed with native PAGE (Fig. 4A) was confirmed by the DNase I binding assay.
44. The crosslinked form (T-TRiC) displayed a faster mobility on native PAGE than did TRiC (Fig. 4B), either because of the added negative charge conferred by the covalently bound nucleotide or because of a conformational change triggered by nucleotide binding.
45. Identical results were obtained for T-GroEL and T-TRiC when actin folding was analyzed as a function of binding to DNase I-Sepharose beads.
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52. We thank M. Wiedmann for pGEM-CAT, J. Shephard for pGEM-mouse β-actin, and M. Mayhew, D. McColl, and B. Netzer for critical reading of the manuscript. Supported by NIH and the Howard Hughes Medical Institute.