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Title: Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins

Abstract

The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads tomore » inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1372257
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry; Journal Volume: 292; Journal Issue: 19
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Tripathi, Arati, Mandon, Elisabet C., Gilmore, Reid, and Rapoport, Tom A. Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins. United States: N. p., 2017. Web. doi:10.1074/jbc.M116.761122.
Tripathi, Arati, Mandon, Elisabet C., Gilmore, Reid, & Rapoport, Tom A. Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins. United States. doi:10.1074/jbc.M116.761122.
Tripathi, Arati, Mandon, Elisabet C., Gilmore, Reid, and Rapoport, Tom A. Sun . "Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins". United States. doi:10.1074/jbc.M116.761122.
@article{osti_1372257,
title = {Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins},
author = {Tripathi, Arati and Mandon, Elisabet C. and Gilmore, Reid and Rapoport, Tom A.},
abstractNote = {The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.},
doi = {10.1074/jbc.M116.761122},
journal = {Journal of Biological Chemistry},
number = 19,
volume = 292,
place = {United States},
year = {Sun Mar 12 00:00:00 EST 2017},
month = {Sun Mar 12 00:00:00 EST 2017}
}
  • A heat shock-resistant mutant of the budding yeast Saccharomyces cerevisiae was isolated at the mutation frequency of 10/sup -7/ from a culture treated with ethyl methane sulfonate. Cells of the mutant are approximately 1000-fold more resistant to lethal heat shock than those of the parental strain. Tetrad analysis indicates that phenotypes revealed by this mutant segregated together in the ratio 2/sup -/:2/sup -/ from heterozygotes constructed with the wild-type strain of the opposite mating type, and are, therefore, attributed to a single nuclear mutation. The mutated gene in the mutant was herein designated hsr1 (heat shock response). The hsr1 allelemore » is recessive to the HSR1/sup +/ allele of the wild-type strain. Exponentially growing cells of hsr1 mutant were found to constitutively synthesize six proteins that are not synthesized or are synthesized at reduced rates in HSR1/sup +/ cells unless appropriately induced. These proteins include one hsp/G/sub 0/-protein (hsp48A), one hsp (hsp48B), and two G/sub 0/-proteins (p73, p56). Heterozygous liploid (hs1/HSR1/sup +/) cells do not synthesize the proteins constitutively induced in hsr1 cells, which suggests that the product of the HSR1 gene might negatively regulate the synthesis of these proteins. The hsr1 mutation also led to altered growth of the mutant cells. The mutation elongated the duration of G/sub 1/ period in the cell cycle and affected both growth arrest by sulfur starvation and growth recovery from it. The authors discuss the problem of which protein(s) among those constitutively expressed in growing cells of the hsr1 mutant is responsible for heat shock resistance and alterations in the growth control.« less
  • The basal promoter of the human gene encoding 70-kDa heat shock protein (HSP70) controls maximal transcriptional activity and serum-regulated expression. The authors demonstrate that the three promoter elements defined by in vivo studies - CCAAT, serum-regulated element (SRE), and TATA - correspond to protein-binding sites in vitro. The promoter interactions with protein factors in HeLa cell crude nuclear extracts were detected by an exonuclease III digestion assay. The sequence specificity was demonstrated with promoter probes containing wild-type sequences or unique linker-scanner mutations that alter each of the elements. They suggest that the protein factor binding to the SRE is involvedmore » in the serum-regulated expression of the human gene for HSP70.« less
  • G{sub o} is a specific class (other) of signal-transducing heterotrimeric GTP-binding proteins (G proteins) that is expressed in high levels in mammalian brain. The authors have cloned two different rat cDNAs encoding the {alpha} subunit of G{sub o} (G{sub o}{alpha}-1 and G{sub o}{alpha}-2) and a human G{sub o}{alpha} chromosomal gene. The human G{sub o}{alpha} gene spans more than 100 kilobases and contains 11 exons, including one noncoding exon in the 3{prime} flanking region. The 5{prime} flanking region is highly G + C-rich and contains five G{center dot}C boxes (Sp1 binding sites) but no TATA box. Exons 7 and 8 codingmore » for amino acid residues 242-354 of G{sub o}{alpha} protein are duplicated. It was found that exons 7A and 8A code for G{sub o}{alpha}01, and 7B and 8B code for G{sub o}{alpha}-2. Sequence homology and conservation of the exon-intron organization indicate that the genes coding for G{sub o}{alpha}, G{sub i}2{alpha}, G{sub i}3{alpha}, G{sub t}1{alpha}, and probably G{sub i}1{alpha} may be evolved from a common progenitor. Like G{sub o}{alpha}-1, G{sub o}{alpha}-2 is expressed mainly in brain.« less
  • Recent interests are beginning to be directed towards toxic neurobiological dysfunctions caused by lead (Pb) in aquatic vertebrates. In the present work, treatment with a maximum acceptable toxic concentration of this heavy metal was responsible for highly significant (p < 0.01) abnormal motor behaviors such as hyperactive movements in the teleost Thalassoma pavo and the same treatment accounted for significantly (p < 0.05) enhanced hyperventilating states. On the other hand, greater abnormal motor behaviors were detected in the presence of the histamine (HA) receptor subtype 2 (H{sub 2}R) antagonist cimetidine (Cim), as shown by the very robust (p < 0.001)more » increases of the two behavioral states. Interestingly, elevated expression levels of stress-related factors, i.e. heat shock protein70/90 (HSP90/70) orthologs were reported for the first time in hypothalamic and mesencephalic areas of Pb-treated teleosts. In particular, an up-regulation of HSP70 was readily detected when this heavy metal was given concomitantly with Cim, while the histamine subtype 3 antagonist (H{sub 3}R) thioperamide (Thio), instead, blocked Pb-dependent up-regulatory trends of both chaperones in mostly hypothalamic areas. Moreover, intense neuronal damages of the above brain regions coincided with altered expressions of HSP70 and HSP90 when treated only with Cim. Overall these first results show that distinct H{sub n}R are able to exert a net neuroprotective role arising from their interaction with chaperones in fish exposed to Pb-dependent stressful conditions making this a potentially key interaction especially for T. pavo, aquatic species which plays an important ecological role towards the survival of other commercially vital fishes.« less
  • Thrombin plays a critical role in platelet activation, hemostasis, and thrombosis. Cellular activation by thrombin leads to the phosphorylation of multiple proteins, most of which are unidentified. The authors have characterized several 29-kDa proteins that are rapidly phosphorylated following exposure of intact human platelets to thrombin. A murine monoclonal antibody raised to an unidentified estrogen receptor-related 29-kDa protein selectively recognized these proteins as well as a more basic, unphosphorylated 27-kDa protein. Cellular activation by thrombin led to a marked shift in the proportion of protein from the 27-kDa unphosphorylated form to the 29-kDa phosphoprotein species. Using this antibody, they isolatedmore » and sequenced a human cDNA clone encoding a protein that was identical to the mammalian 27-kDa heat shock protein (HSP27), a protein of uncertain function that is known to be phosphorylated to several forms and to be transcriptionally induced by estrogen. The 29-kDa proteins were confirmed to be phosphorylated forms of HSP27 by immunoprecipitation studies. Thus, the estrogen receptor-related protein is HSP27, and the three major 20-kDa proteins phosphorylated in thrombin-activated platelets are forms of HSP27. These data suggest a role for HSP27 in the signal transduction events of platelet activation.« less