Dataset Information

A cell-free system for functional studies of small membrane proteins

ABSTRACT: Numerous small proteins have been discovered across all domains of life, among which many are hydrophobic and predicted to localize to the cell membrane. Based on a few that are well-studied, small membrane proteins are regulators involved in various biological processes, such as cell signaling, nutrient transport, drug resistance, and stress response. However, the function of most identified small membrane proteins remains elusive. Their small size and hydrophobicity make protein production challenging, hindering function discovery. Here, we combined a cell-free system with lipid sponge droplets and synthesized small membrane proteins in vitro. Lipid sponge droplets contain a dense network of lipid bilayers, which accommodates and extracts newly synthesized small membrane proteins from the aqueous surroundings. Using small bacterial membrane proteins MgrB, SafA, and AcrZ as proof of principle, we showed that the in vitro produced membrane proteins were functionally active, for example, modulating the activity of their target kinase as expected. Furthermore, small proteins produced in this system could be used for in vitro co-immunoprecipitations to identify interaction partners. The cell-free system produced small membrane proteins, including one from human, up to micromolar concentrations, indicating its high level of versatility. This work presents a robust alternative approach for producing small membrane proteins, which opens a door to their function discovery in different domains of life.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli

SUBMITTER: Timo Glatter

LAB HEAD: Timo Glatter

PROVIDER: PXD051183 | Pride | 2025-05-06

REPOSITORIES: Pride

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A cell-free system for functional studies of small membrane proteins.

Jiang Shan S Çelen Gülce G Glatter Timo T Niederholtmeyer Henrike H Yuan Jing J

The Journal of biological chemistry 20241001 11

Numerous small proteins have been discovered across all domains of life, among which many are hydrophobic and predicted to localize to the cell membrane. Based on a few that are well-studied, small membrane proteins are regulators involved in various biological processes, such as cell signaling, nutrient transport, drug resistance, and stress response. However, the function of most identified small membrane proteins remains elusive. Their small size and hydrophobicity make protein production cha ...[more]

PMID: 39362471

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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.

			Action	DRS
	PhoQIP-3-1.mzid.gz	Mzid
	PhoQIP-3-1.mzid_PhoQIP-3-1.MGF	Mzid
	PhoQIP-3-1.raw	Raw
	PhoQIP-3-2.mzid.gz	Mzid
	PhoQIP-3-2.mzid_PhoQIP-3-2.MGF	Mzid