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Genome-wide identification of bacterial membrane protein expression determinants

ABSTRACT: Cellular membranes provide a barrier to toxins, play a central role in metabolism, and are essential for cell viability, making membrane proteins of key interest for microbial strain engineering. However, membrane protein expression often causes decreased cell viability and alterations to central metabolism. We hypothesized that some non-essential proteins may be antagonistic towards the expression of a desired membrane protein and thus expression stress may be alleviated by the deletion of the corresponding genes. To identify mutant Escherichia coli that tolerate greater expression of membrane proteins, we developed a generalizable method, FlowSeq, that utilizes a pooled, bar-coded transposon insertion library in tandem with cell sorting to access genome-wide impact on membrane protein expression. Five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST) fusted to GFP were screened with FlowSeq. We identified 15 gene deletion strains capable of increased membrane protein expresison, and 3 of these knockout strains also increased membrane protein function. To our knowledge, this study represents the first systematic approach to identify genetic deteminants of membrane protein expression.

ORGANISM(S): Escherichia coli BW25113

PROVIDER: GSE95857 | GEO | 2017/10/03

SECONDARY ACCESSION(S): PRJNA378608

REPOSITORIES: GEO

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Project description:The ARV1-encoded protein mediates sterol transport from the endoplasmic reticulum (ER) to the plasma membrane. Yeast ARV1 mutants accumulate multiple lipids in the ER and are sensitive to pharmacological modulators of both sterol and sphingolipid metabolism. Using fluorescent and electron microscopy, we demonstrate sterol accumulation, subcellular membrane expansion, elevated lipid droplet formation and vacuolar fragmentation in ARV1 mutants. Motif-based regression analysis of ARV1 deletion transcription profiles indicates activation of Hac1p, an integral component of the UPR. Accordingly, we show constitutive splicing of HAC1 transcripts, induction of a UPR reporter and elevated expression of UPR targets in ARV1 mutants. IRE1, encoding the unfolded protein sensor in the ER lumen, exhibits a lethal genetic interaction with ARV1, indicating a viability requirement for the UPR in cells lacking ARV1. Surprisingly, ARV1 mutants expressing a variant of Ire1p defective in sensing unfolded proteins are viable. Moreover these strains also exhibit constitutive HAC1 splicing that interacts with DTT-mediated perturbation of protein folding. These data suggest a component of UPR induction in arv1? strains is distinct from protein misfolding. Decreased ARV1 expression in murine macrophages also results in UPR induction, particularly up-regulation of activating transcription factor-4, C/EBP homologous protein (CHOP) and apoptosis. Cholesterol loading or inhibition of cholesterol esterification further elevated CHOP expression in ARV1 knockdown cells. Thus, loss or down-regulation of ARV1 disturbs membrane and lipid homeostasis resulting in a disruption of ER integrity, one consequence of which is induction of the UPR. Yeast strains were grown to mid-logarithmic stage (A600=0.5-0.6) for RNA extraction and hybridization on Ye6100 or S98 Affymetrix gene chips. The control array (sample name C) was carried out in duplicate. The ARV1 mutant array (sample name A) was carried out in triplicate. Both ARV1 mutants Ye6100 arrays were compared to the same control Ye6100 array. Strains represent different isolates of the same genotype, mating type and genetic background (w303). Sturley lab collection (SCY) strains include SCY328 and SCY2004 for controls and SCY820 and SCY840 for ARV1 mutants.

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Genome-wide identification of bacterial membrane protein expression determinants

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