Dataset Information

BioID performed on Golgi-enriched fractions identify C10orf76 as a GBF1 Binding Protein essential for Golgi maintenance and secretion

ABSTRACT: Data contains LC-MS data of proximity-based biotin labeling assay (BioID) to generate the protein interaction network of GBF1 protein fused to FlagBirA (N-terminal tag) and expressed in HeLa cells and extracted from whole cell lysates and Golgi fractions.

INSTRUMENT(S): Q Exactive HF (Thermo)

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

SUBMITTER: Brian Raught

PROVIDER: MSV000083866 | MassIVE | Mon May 27 18:12:00 BST 2019

SECONDARY ACCESSION(S): PXD014024

REPOSITORIES: MassIVE

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Project description:Protein secretion is an essential process in cell biology. The conventional secretion pathway is well known, and involves proteins with a signal sequence being directed into the endoplasmic reticulum. Once in the lumen of the endoplasmic reticulum, proteins are trafficked through the Golgi and remain separate from the cytosol throughout their journey to the outside of the cell. More recently, proteins without a signal sequence have been found outside the cell. These proteins are secreted unconventionally, and avoid the ER-Golgi route. For both pathways, some aspects remain unclear, particularly surrounding the regulation of protein secretion. Galectin-3, an unconventionally secreted protein, was used here to investigate both conventional and unconventional protein secretion. The unconventional protein secretion of galectin-3 is poorly understood. To address this, genetic manipulation of galectin-3 was used to gain insights into the mechanism of galectin-3 secretion. It was found that galectin-3 does not require unfolding or binding to cell surface glycoproteins to be secreted. Galectin-3 was also used to assess protein secretion using a genome-wide CRISPR screen. As galectin‑3 binds to cell surface glycoproteins, galectin-3 on the cell surface was used to assess secretion of these glycoproteins. Hits were validated by a glycoprotein secretion assay. Using this pipeline, 93 hits were identified as involved in protein secretion, of which 51 were novel hits not previously associated with protein secretion. Many of the hits identified also resulted in a phenotype of altered Golgi morphology, and five of these were investigated further. Two novel hits that localised to the Golgi were identified, TMEM220 and GPR161. GPR161 likely mediates its effect on Golgi morphology by its interaction with golgin A5. This thesis presents new insights into both conventional and unconventional protein secretion. Insights into the mechanism for galaectin-3 secretion were revealed. A novel Golgi-localised regulator of secretion, GRP161, was identified, with a mechanism for action suggested. Importantly, the list of 51 novel genes identified will serve as a useful resource for other researchers investigating protein secretion.

Dataset Information

BioID performed on Golgi-enriched fractions identify C10orf76 as a GBF1 Binding Protein essential for Golgi maintenance and secretion

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