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Protein partners identification in Pyrococcus abyssi by nanoLC-MSMS

ABSTRACT: Recombinant aRNase J from P. abyssi, with C-terminal (His)6-tag (aRNase J-His), was produced, purified and used as baits for AP-MS experiments. In order to discriminate specific protein interaction from column background, we performed mock AP-MS analyses with P. abyssi cell extracts in absence of protein bait. In addition, to determine if some interactions are mediated through RNA or DNA, we implemented a nuclease treatment after incubating the cell extract with the bait. The co-purified partners were identified by bottom-up proteomic techniques coupled with mass spectrometry.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Pyrococcus Abyssi

SUBMITTER: Lydie Correia

LAB HEAD: Lydie Oliveira Correia

PROVIDER: PXD015856 | Pride | 2020-01-21

REPOSITORIES: Pride

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Project description:During lactation, mammary epithelial secretory cells secrete huge amounts of milk from their apical side. The major milk proteins, the caseins, are secreted by exocytosis, while milk fat globules are released by budding, enwrapped by the plasma membrane. Due to the number and large size of milk fat globules, the membrane surface needed for their secretion might exceed that of the apical plasma membrane. In order to identify the cellular compartments that may provide membrane during the budding of milk fat globules, a large-scale proteomics analysis of both cytoplasmic lipid droplets and secreted milk fat globules membranes was performed in mouse cells. The differential analysis of the protein profiles of these two organelles strongly suggest that, in addition to the apical plasma membrane, at least the endoplasmic reticulum, the secretory vesicles containing caseins, and potentially the mitochondria contribute to the formation of the milk fat globule membrane. Moreover, the specific analysis of the membrane-associated as well as the raft-associated proteins reinforces this possibility and points to a role for lipid rafts in milk product secretion. The subcellular localization of several major proteins of the cytoplasmic lipid droplets or of the milk fat globule membrane was investigated by immunofluorescence in lactating mammary epithelial cells. Furthermore, the localization of GM1 ganglioside, a known marker of lipid rafts and of free cholesterol, showed a clear association with both cytoplasmic lipid droplets and secreted milk fat globules. Additionally, the presence of some SNARE proteins that may be involved in casein exocytosis and of two Rab GTPases on both cytoplasmic lipid droplets and milk fat globules was demonstrated by immunofluorescence. Altogether, our results provide evidence for a pivotal role of the endoplasmic reticulum as membrane contributor to milk fat globule budding, and suggest that some SNARE proteins may spatio-temporally coordinate the secretion of milk products.

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Protein partners identification in Pyrococcus abyssi by nanoLC-MSMS

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