Dataset Information

Targeting of precursor polypeptides to the human ER

ABSTRACT: Various pathways can target nascent or fully synthesized precursor polypeptides to the human endoplasmic reticulum (ER). Typically, they involve cytosolic proteins or complexes and their respective receptors on the ER surface. The signal recognition particle (SRP) and the heterodimeric SRP-receptor (SR) represent one such targeting system, others are TRC40 and its heterodimeric TRC-receptor (WRB/CAML) and the components of the SND pathway. Apparently, they all can target precursor polypeptides to the Sec61-channel in the ER membrane. To characterize the substrate specificities of these targeting pathways, we combined siRNA-mediated depletion of membrane receptor subunits in HeLa cells with label-free quantitative proteomics and differential protein abundance analyis.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Richard Zimmermann

LAB HEAD: Prof. Dr. Richard Zimmermann (lab head)

PROVIDER: PXD011993 | Pride | 2020-03-06

REPOSITORIES: Pride

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Publications

Identification of signal peptide features for substrate specificity in human Sec62/Sec63-dependent ER protein import.

Schorr Stefan S Nguyen Duy D Haßdenteufel Sarah S Nagaraj Nagarjuna N Cavalié Adolfo A Greiner Markus M Weissgerber Petra P Loi Marisa M Paton Adrienne W AW Paton James C JC Molinari Maurizio M Förster Friedrich F Dudek Johanna J Lang Sven S Helms Volkhard V Zimmermann Richard R

The FEBS journal 20200320 21

In mammalian cells, one-third of all polypeptides are integrated into the membrane or translocated into the lumen of the endoplasmic reticulum (ER) via the Sec61 channel. While the Sec61 complex facilitates ER import of most precursor polypeptides, the Sec61-associated Sec62/Sec63 complex supports ER import in a substrate-specific manner. So far, mainly posttranslationally imported precursors and the two cotranslationally imported precursors of ERj3 and prion protein were found to depend on the ...[more]

PMID: 32133789

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Project description:Glioblastoma multiforme (GBM) is the most prevalent type of adult brain tumor, and one of the deadliest tumors known to mankind. The genetic understanding of GBM is, however, limited, and the molecular mechanisms which facilitate GBM cell survival and growth within the tumor microenvironment are largely unknown. We applied digital karyotyping and single nucleotide polymorphism (SNP) arrays to screen for copy number changes in GBM samples and found that the most frequently amplified region is at chromosome 7p11.2. The high resolution of digital karyotyping and SNP arrays permits the precise delineation of amplicon boundaries and has enabled identification of the minimal region of amplification at 7p11.2, which contains two genes, EGFR and SEC61?. SEC61? encodes a subunit of a heterotrimeric protein channel located in the endoplasmic reticulum (ER). In addition to its high frequency of gene amplification in GBMs, SEC61? is also remarkably overexpressed in 77% of GBMs, but not in lower-grade gliomas. The siRNA-mediated knockdown of SEC61? expression in tumor cells led to growth suppression and apoptosis. Furthermore, we showed that pharmacological ER stress agents induce SEC61? expression in GBM cells. Together, these results indicate that aberrant expression of SEC61? serves significant roles in GBM cell survival, likely via a mechanism that is involved in the cytoprotective ER stress-adaptive response to the tumor microenvironment. hSETD1A was silenced in HCT116 cells using retrovirus harboring shRNA specifically against the hSETD1A gene. 10?g RNA was reverse transcribed to cDNA using the Applied Biosystems High Capacity cDNA Reverse Transcription kit according to the manufacturer’s instructions (Applied Biosystems). cDNA products were treated with 100ng RNaseA and then purified using the Qiagen PCR purification kit according to the manufacturer’s instructions. NimbleGen Human Gene Expression array was purchased from Roche Applied Sciences. cDNA samples were labeled, Cy3 hybridized, and processed at the FSU NimbleGen Microarray Facility at Florida State University.

Dataset Information

Targeting of precursor polypeptides to the human ER

Publications

Identification of signal peptide features for substrate specificity in human Sec62/Sec63-dependent ER protein import.

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