Dynamic localization of membrane proteins in Bacillus subtilis

Abstract

The subcellular localization of membrane proteins in Bacillus subtilis was examined by using fluorescent protein fusions. ATP synthase and succinate dehydrogenase were found to localize within discrete domains on the membrane rather than being homogeneously distributed around the cell periphery as expected. Dual labelling of cells indicated partial colocalization of ATP synthase and succinate dehydrogenase. Further analysis using an ectopically expressed phage protein gave the same localization patterns as ATP synthase and succinate dehydrogenase, implying that membrane proteins are restricted to domains within the membrane. 3D reconstruction of images of the localization of ATP synthase showed that domains were not regular and there was no bias for localization to cell poles or any other positions. Further analysis revealed that this localization was highly dynamic, but random, implying that integral membrane proteins are free to diffuse two-dimensionally around the cytoplasmic membrane.

Characterization and subcellular localization of a bacterial flotillin homologue

“…Probably, the localization of FtsH to sites with negatively charged lipids results in the isolation of a fraction of FtsH in the low-density fractions. ATP synthase is localized within the entire cytoplasmic membrane and is therefore unlikely to be concentrated in lipid microdomains (Johnson et al, 2004).…”

Different patterns of integral membrane protein localization during cell division in Bacillus subtilis

“…1A). The pattern of localization for both SdhA and AtpA fusions was heterogeneous, with multiple small regions of fluorescence of varying levels of intensity (see Johnson et al, 2004). Due to this highly varied level of fluorescence around the cytoplasmic membrane, no obvious pattern of low signal at mid-cell was apparent in either of the single-labelled strains.…”

“…Cyan fluorescent protein (CFP) fluorescence was visualized with filter set 31044v2, green fluorescent protein (GFP) with set 41018, and yellow fluorescent protein (YFP) with set 41029 (Chroma Technology). Image processing and analysis was performed as described by Johnson et al (2004) bla amyE39 spc P xyl -p16.7-cfp amyE59 This work, PCR p16.7 from BS125 and insert into pSG1192 pNG534 bla amyE39 spc P xyl -gfp-secDF amyE59 This work, secDF inserted into pSG1729 one of the cell poles. In the resulting intensity profile, the mid-cell sites would therefore correspond approximately to the one-quarter and three-quarter points on the x-axes.…”

“…2A; Table 1). p16.7 is an integral membrane protein from the B. subtilis phage w29 that is involved in phage DNA replication, and has previously been shown to localize around the cell membrane when ectopically expressed (Meijer et al, 2001;Johnson et al, 2004). This protein plays no role in the metabolism of uninfected B. subtilis and so was used to determine if the appearance of CRs was a non-specific effect on integral membrane proteins at the onset of cell division.…”

“…In previous studies we investigated the distribution of succinate dehydrogenase and ATP synthase complexes using fluorescent protein fusions to the AtpA and SdhA subunits, respectively, which showed that both complexes were distributed around the cytoplasmic membrane (Johnson et al, 2004). The expression of both fluorescent fusions was driven by the activity of the wild-type promoters, and polar effects of insertion of the fusions into the chromosome were avoided through expression of downstream genes from the xylose-inducible promoter (see Johnson et al, 2004 and Fig.…”