Hsp70 forms antiparallel dimers stabilized by post-translational modifications to position clients for transfer to Hsp90.
Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90.
Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK. Electronic address: morgner@chemie.uni-frankfurt.de.
Nina
N
Morgner
Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.
Carla
C
Schmidt
Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.
Victoria
V
Beilsten-Edmands
Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.
Ima-Obong
IO
Ebong
Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.
Nisha A
NA
Patel
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA 01003, USA.
Eugenia M
EM
Clerico
Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA.
Elaine
E
Kirschke
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
Soumya
S
Daturpalli
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
Sophie E
SE
Jackson
Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA.
David
D
Agard
Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK. Electronic address: carol.robinson@chem.ox.ac.uk.
Carol V
CV
Robinson
D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D050956:HSP40 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D017510:Protein Folding; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins