Dataset Information

A novel translocon for biogenesis of multi-pass membrane proteins

ABSTRACT: A novel 370 kDa translocon comprising Sec61 and five accessory factors acting co-translationally with ribosomes to fold and insert multi-pass membrane proteins was identified, purified and analyzed structurally by single particle cryo-EM and cross-linking mass spectrometry. The purified ribosome-translocon complex was cross-linked with DSS, trypsin digested, and size-exclusion enriched (SEC) cross-link containing fractions were analyzed by sequential HCD/ETD of the same precursor ion. The four LC-MS runs corresponding to SEC fractions eluting between 0.9-1.4 ml are deposited here.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Robert J. Keenan

PROVIDER: MSV000084065 | MassIVE | Mon Jul 08 11:56:00 BST 2019

REPOSITORIES: MassIVE

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Publications

An ER translocon for multi-pass membrane protein biogenesis.

McGilvray Philip T PT Anghel S Andrei SA Sundaram Arunkumar A Zhong Frank F Trnka Michael J MJ Fuller James R JR Hu Hong H Burlingame Alma L AL Keenan Robert J RJ

eLife 20200821

Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here we describe a ~ 360 kDa ribosome-associated complex comprising the core Sec61 channel and five accessory factors: TMCO1, CCDC47 and the Nicalin-TMEM147-NOMO complex. Cryo-electron microscopy reveals a large assembly at the ribosome exit tunnel organized around a central membrane cavity. Similar to pr ...[more]

PMID: 32820719

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Project description:In the marrow and lymphatic tissues, chronic lymphocytic leukemia (CLL) cells interact with accessory cells that constitute the leukemia microenvironment. In lymphatic tissues, CLL cells are interspersed with CD68+ nurselike cells (NLC) and T cells. However, the mechanism regulating co-localization of CLL cells and these accessory cells are largely unknown. To dissect the molecular cross-talk between CLL and NLC, we profiled the gene expression of CD19-purified CLL cells before and after co-culture with NLC. NLC co-culture induced high-level expression of B cell maturation antigen (BCMA) and two chemoattractants (CCL3, CCL4) by CLL cells. Supernatants from CLL-NLC co-cultures revealed high CCL3/CCL4 protein levels. B cell receptor triggering also induced a robust induction of CCL3 and CCL4 expression by CLL cells, which was almost completely abrogated by a specific Syc inhibitor, R406. High CCL3 and CCL4 plasma levels in CLL patients suggest that activation of this pathway plays a role in vivo. These studies reveal a novel mechanism of cross-talk between CLL cells and their microenvironment, namely the secretion of two T cell chemokines by CLL-NLC interaction and in response to BCR stimulation. Through these chemokines, CLL cells can recruit accessory cells, and thereby actively create a microenvironment that favors their growth and survival. Experiment Overall Design: The microarray part of this study included samples of CLL cells from 9 different patients, analyzed directly after purification and after 14 days of co-culture with Nurse like cells . Experiment Overall Design: In detail, RNA was isolated from CD19-purified CLL cells from 9 different patientsâ peripheral blood mononuclear cells (PBMC) after Ficoll separation and subsequent purification with CD19 MicroBeads and the MACSÂ® technology according to the manufacturerâs instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). For comparison, the same CLL cell samples were co-cultured for 14 days with NLC (""14d NLC"").""). For co-culture with NLC, PBMC from patients with CLL were suspended in complete RPMI medium (RPMI1640 with 10% FCS, penicillin-streptomycin-glutamine, Gibco-BRL, Grand Island, NY) to a concentration of 1 x 107/ml (total 20 ml) and incubated for 14 days in 75 cm2 tissue culture flasks (Techno Plastic Products AG) as described previously3. Nonadherent lymphoid cells then were removed and the NLC layer was washed two times with phosphate-buffered saline (PBS). The complete removal of lymphocytes was verified by phase-contrast microscopy. The nonadherent cells together with the wash-fractions were then used for RNA preparation. In order to purify the CLL B cells prior to RNA isolation, CLL PBMC were passed through a 30 Âµm nylon mesh to obtain a single-cell suspension. Then CLL B cells were purified with CD19 MicroBeads. Subsequently RNA was extracted.

Dataset Information

A novel translocon for biogenesis of multi-pass membrane proteins

Publications

An ER translocon for multi-pass membrane protein biogenesis.

Similar Datasets

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