Subcellular localization of Escherichia coli osmosensory transporter ProP: focus on cardiolipin membrane domains (original) (raw)
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Cardiolipin promotes polar localization of osmosensory transporter ProP in Escherichia coli
Molecular Microbiology, 2007
The osmolality required to activate osmosensory transporter ProP and the proportion of cardiolipin (CL) among the phospholipids of Escherichia coli rise with growth medium osmolality. Most CL synthesis has been attributed to the cls gene product. Transcription of cls increased with osmolality. The proportion of CL was low and osmolality-independent in cls(-) bacteria. It increased more dramatically on the transition to stationary phase in cls(-) than cls(+) bacteria. Thus, Cls is responsible for osmoregulated CL synthesis and other enzymes may contribute to CL accumulation during stationary phase. The proportion of phosphatidylglycerol (PG) was elevated and it increased with medium osmolality in cls(-) bacteria. A cls defect impaired growth of E. coli on solid and in liquid media at low and, more strongly, at high osmolality. Bacteria cultured at high osmolality without osmoprotectant were shorter and rounder than those cultured at low osmolality or with glycine betaine. Fluorescence microscopy showed that CL and ProP colocalize at the poles and near the septa of dividing E. coli cells. The polar localization of ProP was independent of its expression level but correlated with the proportion and polar localization of CL. Association with CL (and not PG) may be required for polar ProP localization.
Molecular microbiology, 2017
Osmosensing by transporter ProP is modulated by its cardiolipin (CL)-dependent concentration at the poles of Escherichia coli cells. Other contributors to this phenomenon were sought with the BACterial Two-Hybrid System (BACTH). The BACTH-tagged variants T18-ProP and T25-ProP retained ProP function and localization. Their interaction confirmed the ProP homo-dimerization previously established by protein crosslinking. YdhP, YjbJ and ClsA were prominent among the putative ProP interactors identified by the BACTH system. The functions of YdhP and YjbJ are unknown, although YjbJ is an abundant, osmotically induced, soluble protein. ClsA (CL Synthase A) had been shown to determine ProP localization by mediating CL synthesis. Unlike a deletion of clsA, deletion of ydhP or yjbJ had no effect on ProP localization or function. All three proteins were concentrated at the cell poles, but only ClsA localization was CL-dependent. ClsA was shown to be N-terminally processed and membrane-anchored,...
Biochemistry, 1999
The ProP protein of Escherichia coli is an osmoregulatory H +-compatible solute cotransporter. ProP is activated by an osmotic upshift in both whole cells and membrane vesicles. We are using biochemical and biophysical techniques to explore the osmosensory and catalytic mechanisms of ProP. We now report the purification and reconstitution of the active transporter. Protein purification was facilitated by the addition of six histidine (His) codons to the 3′ end of proP. The recombinant gene was overexpressed from the E. coli galP promoter, and ProP-(His) 6 was shown to be functionally equivalent to wild-type ProP by enzymatic assay of whole cells. ProP-(His) 6 , purified by Ni 2+ (NTA) affinity chromatography, cross-reacted with antibodies raised against the ProP protein. ProP-(His) 6 was reconstituted into Triton X-100 destabilized liposomes prepared with E. coli phospholipid. The reconstituted transporter mediated proline accumulation only if (1) a membrane potential was generated by valinomycinmediated K + efflux and (2) the proteoliposomes were subjected to an osmotic upshift (0.6 M sucrose). Activity was also stimulated by ∆pH. Pure ProP acts, in the proteoliposome environment, as sensor, transducer, and respondent to a hyperosmotic shift. It is the first such osmosensor to be isolated.
Cardiolipin and the osmotic stress responses of bacteria
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2009
Cells control their own hydration by accumulating solutes when they are exposed to high osmolality media and releasing solutes in response to osmotic down-shocks. Osmosensory transporters mediate solute accumulation and mechanosensitive channels mediate solute release. Escherichia coli serves as a paradigm for studies of cellular osmoregulation. Growth in media of high salinity alters the phospholipid headgroup and fatty acid compositions of bacterial cytoplasmic membranes, in many cases increasing the ratio of anionic to zwitterionic lipid. In E. coli, the proportion of cardiolipin (CL) increases as the proportion of phosphatidylethanolamine (PE) decreases when osmotic stress is imposed with an electrolyte or a nonelectrolyte. Osmotic induction of the gene encoding CL synthase (cls) contributes to these changes. The proportion of phosphatidylglycerol (PG) increases at the expense of PE in cls − bacteria and, in Bacillus subtilis, the genes encoding CL and PG synthases (clsA and pgsA) are both osmotically regulated. CL is concentrated at the poles of diverse bacterial cells. A FlAsH-tagged variant of osmosensory transporter ProP is also concentrated at E. coli cell poles. Polar concentration of ProP is CL-dependent whereas polar concentration of its paralogue LacY, a H + -lactose symporter, is not. The proportion of anionic lipids (CL and PG) modulates the function of ProP in vivo and in vitro. These effects suggest that the osmotic induction of CL synthesis and co-localization of ProP with CL at the cell poles adjust the osmolality range over which ProP activity is controlled by placing it in a CL-rich membrane environment. In contrast, a GFP-tagged variant of mechanosensitive channel MscL is not concentrated at the cell poles but anionic lipids bind to a specific site on each subunit of MscL and influence its function in vitro. The sub-cellular locations and lipid dependencies of other osmosensory systems are not known. Varying CL content is a key element of osmotic adaptation by bacteria but much remains to be learned about its roles in the localization and function of osmoregulatory proteins.
Scientific Reports, 2020
Translocation of preproteins across the Escherichia coli inner membrane requires anionic lipids by virtue of their negative head-group charge either in vivo or in situ. However, available results do not differentiate between the roles of monoanionic phosphatidylglycerol and dianionic cardiolipin (CL) in this essential membrane-related process. To define in vivo the molecular steps affected by the absence of CL in protein translocation and insertion, we analyzed translocon activity, SecYEG stability and its interaction with SecA in an E. coli mutant devoid of CL. Although no growth defects were observed, co- and post-translational translocation of α-helical proteins across inner membrane and the assembly of outer membrane β-barrel precursors were severely compromised in CL-lacking cells. Components of proton-motive force which could impair protein insertion into and translocation across the inner membrane, were unaffected. However, stability of the dimeric SecYEG complex and oligomer...
Biochemistry, 2003
Transporter ProP of Escherichia coli is an osmosensor and an osmoprotectant transporter. Previous results suggest that medium osmolality determines the proportions of ProP in active and inactive conformations. A cysteine-less (Cys-less) variant was created and characterized as a basis for structural and functional analyses based on site-directed Cys substitution and chemical labeling of ProP. Parameters describing the osmosensory and osmoprotectant transport activities of Cys-less ProP-(His) 6 variants were examined, including the threshold for osmotic activation and the absolute transporter activity at high osmolality (in both cells and proteoliposomes), the dependence of K M and V max for proline uptake on osmolality, and the rate constant for transporter activation in response to an osmotic upshift (in cells only). Variant ProP-(His) 6 -C112A-C133A-C264V-C367A (designated ProP*) retained similar activities to ProP-(His) 6 in both cells and proteoliposomes. The bulky Val residue was favored over Ala or Ser at position 264, whereas Val strongly impaired function when placed at position 367, highlighting the importance of residues at those positions for osmosensing. In the ProP* background, variants with a single Cys residue at positions 112, 133, 241, 264, 293, or 367 retained full function. The native Cys at positions 112, 133, 264, and 367, predicted to be within transmembrane segments of ProP, were poorly reactive with membrane-impermeant thiol reagents. The reactivities of Cys at positions 241 and 293 were consistent with exposure of those residues on the cytoplasmic and periplasmic surfaces of the cytoplasmic membrane, respectively. These observations are consistent with the topology and orientation of ProP predicted by hydropathy analysis.