Unbalanced charge distribution as a determinant for dependence of a subset of Escherichia coli membrane proteins on the membrane insertase YidC

Abstract

Membrane proteins are involved in numerous essential cell processes, including transport, gene regulation, motility, and metabolism. To function properly, they must be inserted into the membrane and folded correctly. YidC, an essential protein in Escherichia coli with homologues in other bacteria, Archaea, mitochondria, and chloroplasts, functions by incompletely understood mechanisms in the insertion and folding of certain membrane proteins. Using a genome-scale approach, we identified 69 E. coli membrane proteins that, in the absence of YidC, exhibited aberrant localization by microscopy. Further examination of a subset revealed biochemical defects in membrane insertion in the absence of YidC, indicating their dependence on YidC for proper membrane insertion or folding. Membrane proteins possessing an unfavorable distribution of positively charged residues were significantly more likely to depend on YidC for membrane insertion. Correcting the charge distribution of a charge-unbalanced YidC-dependent membrane protein abrogated its requirement for YidC, while perturbing the charge distribution of a charge-balanced YidC-independent membrane protein rendered it YidC dependent, demonstrating that charge distribution can be a necessary and sufficient determinant of YidC dependence. These findings provide insights into a mechanism by which YidC promotes proper membrane protein biogenesis and suggest a critical function of YidC in all organisms and organelles that express it.

Importance: Biological membranes are fundamental components of cells, providing barriers that enclose the cell and separate compartments. Proteins inserted into biological membranes serve critical functions in molecular transport, molecular partitioning, and other essential cell processes. The mechanisms involved in the insertion of proteins into membranes, however, are incompletely understood. The YidC protein is critical for the insertion of a subset of proteins into membranes across an evolutionarily wide group of organisms. Here we identify a large group of proteins that depend on YidC for membrane insertion in Escherichia coli, and we identify unfavorable distribution of charge as an important determinant of YidC dependence for proper membrane insertion.

FIG 1

YidC-dependent localization of a subset of GFP-tagged membrane proteins. (a) Seven examples of membrane proteins that showed altered GFP signals in the absence of YidC (− YidC). The AtpB protein previously shown to be dependent on YidC for membrane insertion (◊) is shown. (b) Membrane protein that showed comparable circumferential GFP signals in the presence (+) and absence (−) of YidC (YaiZ). Images are representative. Bar, 5 µm.

FIG 2

Membrane proteins mislocalized in the absence of YidC are YidC dependent for membrane insertion. (A and B) Proteinase K accessibility assay of spheroplasts depleted of YidC or not depleted of YidC (see Materials and Methods). (A) Experimental approach. (B) Screen hits that displayed reduced protease accessibility following YidC depletion. The spheroplasts were treated with proteinase K (PK) (+) in the protease accessibility assay. Cells pretreated with Triton X-100 (+) to promote lysis served as a control for proteolysis. In the absence of YidC (−), increased abundance of full-length protein (*) and decreased abundance of major cleavage product(s) (**) was observed. The positions of molecular mass standards (in kilodaltons) are shown to the left of the gels. (C to E) Differential fractionation of screen hits into Triton X-100-soluble and -insoluble membrane fractions following synthesis in cells depleted of YidC or not depleted of YidC. (C) Experimental approach. (D) Screen hits that displayed reduced Triton X-100 solubility when synthesized in the absence of YidC. (E) A screen nonhit that displayed comparable Triton X-100 solubilities when synthesized in the presence and absence of YidC. Western blots using antibody to GFP are shown. The cell fractions are as follows: crude, whole-cell proteins; soluble, soluble cytoplasmic and periplasmic proteins; insoluble, membranes and other insoluble proteins; Triton-soluble, Triton X-100-soluble fraction of insoluble proteins; Triton-insoluble, Triton X-100-insoluble fraction of insoluble proteins. The loads were proportional and normalized to the optical density at 600 nm (OD₆₀₀) of culture. Percent solubilization, the ratio of Triton-soluble band to crude fraction band as determined by band densitometry. Images are representative.

FIG 3

The distribution of positively charged residues determines the dependence of CrcB on YidC for membrane insertion. (A) Topological illustrations of the cytoplasmic membrane protein CrcB (22) and variants with altered charge balances. Balanced transmembrane segments (green), charge-neutral transmembrane segments (grey), and charge-unbalanced transmembrane segments (red) are shown. Charge balance altered by mutagenesis is indicated by an asterisk. (B) Subcellular localization of GFP-tagged CrcB (CrcB-GFP) variants in the presence or absence of YidC. Bar, 5 µm. (C and D) Protease susceptibility (C) or differential fractionation into Triton X-100-soluble and -insoluble membrane fractions (D) of CrcB-GFP variants following synthesis in the presence or absence of YidC (see Fig. 2 legend and Materials and Methods). The position of a proteolytic product detected only in the absence of YidC for CrcB_WT but in increasing amounts in the absence of YidC for the balanced variants is indicated by an asterisk. Images are representative; all images in each panel are from the same Western blot or same microscopy experiment.

FIG 4

The distribution of positively charged residues is sufficient to determine the dependence of YaiZ on YidC for membrane insertion. (A) Topological illustrations of the cytoplasmic membrane protein YaiZ (22) and variants with altered charge balances (see Fig. 3 legend). (B) Subcellular localization of YaiZ-GFP variants in the presence or absence of YidC. (C) Differential fractionation of YaiZ-GFP variants into Triton X-100-soluble and -insoluble membrane fractions following synthesis in the presence or absence of YidC (see Fig. 2 legend and Materials and Methods). Images are representative.

FIG 5

Model for YidC-dependent insertion of membrane proteins containing unbalanced transmembrane segments. Transmembrane segments (TMs) of nascent cytoplasmic membrane proteins targeted to the Sec translocon are sequentially inserted into the translocon and then partitioned into the lipid bilayer via a lateral gate. YidC, docked at the lateral gate, interacts with transmembrane segments as they exit the translocon, allowing the stabilization of unbalanced transmembrane segments and folding of the protein with correct topology into an intrinsically stable conformation, which is then released into the lipid bilayer. Balanced transmembrane segments (green) and charge-unbalanced transmembrane segments (red) are shown. Positively charged residues within the flanking extramembrane domain are indicated by plus signs.