Abstract
BackgroundProteins within the cytoplasmic membrane display distinct localization patterns and arrangements. While multiple models exist describing the dynamics of membrane proteins, to date, there have been few systematic studies, particularly in bacteria, to evaluate how protein size, number of transmembrane domains, and temperature affect their diffusion, and if conserved localization patterns exist.ResultsWe have used fluorescence microscopy, single-molecule tracking (SMT), and computer-aided visualization methods to obtain a better understanding of the three-dimensional organization of bacterial membrane proteins, using the model bacterium Bacillus subtilis. First, we carried out a systematic study of the localization of over 200 B. subtilis membrane proteins, tagged with monomeric mVenus-YFP at their original gene locus. Their subcellular localization could be discriminated in polar, septal, patchy, and punctate patterns. Almost 20% of membrane proteins specifically localized to the cell poles, and a vast majority of all proteins localized in distinct structures, which we term microdomains. Dynamics were analyzed for selected membrane proteins, using SMT. Diffusion coefficients of the analyzed transmembrane proteins did not correlate with protein molecular weight, but correlated inversely with the number of transmembrane helices, i.e., transmembrane radius. We observed that temperature can strongly influence diffusion on the membrane, in that upon growth temperature upshift, diffusion coefficients of membrane proteins increased and still correlated inversely to the number of transmembrane domains, following the Saffman–Delbrück relation.ConclusionsThe vast majority of membrane proteins localized to distinct multimeric assemblies. Diffusion of membrane proteins can be suitably described by discriminating diffusion coefficients into two protein populations, one mobile and one immobile, the latter likely constituting microdomains. Our results show there is high heterogeneity and yet structural order in the cell membrane, and provide a roadmap for our understanding of membrane organization in prokaryotes.
Highlights
Proteins within the cytoplasmic membrane display distinct localization patterns and arrangements
A majority of B. subtilis membrane proteins localizes to defined structures within the membrane, termed “microdomains” To study the localization of membrane proteins, we constructed translational fusions of the gene for the Venus yellow fluorescent protein (Venus-YFP) to the 3′ end of genes coding for membrane proteins in the B. subtilis chromosome
Plasmids were integrated into the original gene locus on the B. subtilis chromosome, generating C-terminal membrane protein–monomeric Venus YFP (mVenus) fusions expressed under control of the original promoter as sole source of the protein in the cell
Summary
Proteins within the cytoplasmic membrane display distinct localization patterns and arrangements. While multiple models exist describing the dynamics of membrane proteins, to date, there have been few systematic studies, in bacteria, to evaluate how protein size, number of transmembrane domains, and temperature affect their diffusion, and if conserved localization patterns exist. While initially conceived as a homogeneous lipid bilayer serving as a reaction platform for freely diffusing proteins, the cytoplasmic membrane is widely accepted as being a highly dynamic and compartmentalized environment characterized by heterogeneous diffusion and the presence of microdomains, allowing lipids and proteins to be organized in specific regions of varying size and composition [2, 5,6,7]. Several B. subtilis membrane proteins have been shown to cluster into structures of 60 to 110 nm, supporting evidence for the existence of defined-size protein microdomains [26]
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