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Functional characterization of the ribosomal tunnel exit ligand ERj1

Subject Area Biochemistry
Term from 2008 to 2015
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 50070218
 
Final Report Year 2014

Final Report Abstract

Protein transport into the endoplasmic reticulum (ER) is based on amino-terminal signal peptides in the precursors and the protein translocon comprising the central Sec61 complex in the ER membrane, which forms the polypeptide-conducting channel and for cotranslationally acting translocons the ribosome receptor on the ER membrane. A variety of additional factors including precursor polypeptide processing factors like OST or accompanying factors like molecular chaperones of the Hsp70 and Hsp40 protein families enable the proper transport of thousands of different proteins and were the focus of this collaborative project. Structural in situ insights in the mammalian translocon were obtained using cryo-electron microscopy single-particle analysis and cryo-electron tomography in combination with RNAi technology revealing a detailed picture of the overall spatial organization of the native translocon including the Sec61, TRAP and OST complexes. The precise positioning of the obviously monomeric OST complex on the cotranslationally acting Sec61 translocon allowed conclusions about the molecular mechanism of cotranslational N- glycosylation. The basis for our investigations on the ER membrane Hsp40 protein ERj1 was our previous observations that ERj1 simultaneously interacts with ribosomes in the cytosol and with BiP in the ER lumen. To gain new insights into these interactions, we carried out SPR spectroscopy measurements and determined the apparent affinity of ERj1 for ribosomes (KD 8 nM). The simultaneous binding of BiP reduces the ERj1 affinity for ribosomes. The analog cytosolic Hsp40/Hsp70 pair MPP11/Hsp70L1 (mRAC) showed a similar regulatory behavior at the ribosome. However, MPP11 and mRAC did not compete for ribosome binding of ERj1 indicating that they do not use the same binding site. In the course of this joint project, we identified the human ER membrane protein Sec62 as a novel ribosomal ligand (KD 0.13 nM) with ERj1 overlapping binding sites at the ribosome. Both proteins utilize basic oligopeptide motifs for ribosome binding and bind to nascent chains. Moreover, we showed a Ca2+-sensitive interaction of Sec62 with the Sec61 complex and a direct interaction with the ER membrane Hsp40, Sec63. Using the RNAi technology, we characterized mammalian Sec62 and Sec63 as auxiliary components of the Sec61 complex for ER protein import. Both are involved in the posttranslational import of small proteins as well as of larger transport substrates. Surprisingly, these large Sec62/Sec63-dependent transport substrates were imported SRP-independently into the ER suggesting Sec62/Sec63 being part of an alternative precursor-specific ER membrane targeting system. In line with this we found a precursor-specific involvement of the mammalian Sec63 in the initial phase of cotranslational protein transport into the ER. We characterized the BiP nucleotide exchange factor Sil1 as essential for muscle function in mouse, which makes the woozy mouse carrying a Sil1 gene mutation a suitable in vivo model for the human disorder Marinesco-Sjögren syndrome. Of further medical relevance are our studies on Sec63 and Sec62. We identified the cytosolic protein nucleoredoxin as an interaction partner of human Sec63. Nucleoredoxin is known to be involved in redox-sensitive Wnt signaling pathways, which were linked to cystogenesis. We suggest that the interaction between human Sec63 and nucleoredoxin may contribute to the pathogenesis of the autosomal dominant polycystic liver disease. Our analyses identified SEC62 as a novel oncogene in prostate and lung cancer. We characterized Sec62 as a possible diagnostic and prognostic marker for lung cancer. Besides its influence on cell migration, we showed that tumor cells with a high Sec62 protein content are more resistant against thapsigargin-induced ER stress. The calmodulin antagonist trifluoperacine influences cell migration as well as thapsigargin-induced ER stress tolerance. This provides a novel personalized therapeutic concept for patients with Sec62 overproducing tumors.

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