Mechanosensitive channel functions to alleviate the cell lysis of marine bacterium, Vibrio alginolyticus, by osmotic downshock.
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The mechanosensitive channel with large conductance of Escherichia coli is the first to be cloned among stretch-activated channels. Although its activity was characterized by a patch clamp method, a physiological role of the channel has not been proved. The marine bacterium, Vibrio alginolyticus, is sensitive to osmotic stress and cell lysis occurs under osmotic downshock. We introduced an mscL gene into Vibrio alginolyticus, and the mechanosensitive channel with large conductance functions was found to alleviate cell lysis by osmotic downshock. This is the first report to show a physiological role of the mechanosensitive channel with large conductance. (+info)
Xf phage invading the host cells with their protein coats.
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The Xf phage coat protein associated with infected cells could not be removed by washing with antiserum and tris-EDTA buffer. Although the infected cells were consecutively washed 6 times with tris-EDTA buffer, the ratios of parental phage 6H-DNA to 14C-protein were not changed. A considerable amount of the parental 14C-protein and 3H-DNA in the original ratio were detected in the membrane and the soluble cytoplasmic fractions of infected cells. The studies of the change in Xf 14C-protein and 3H-DNA incorporation into the host cells and their release showed that DNA and protein penetrate together into the host cells during the first 60 min after infection (p.i.). While virtually all parental DNA was conserved, re-utilized and released from the infected cell 60 min p.i., no apparent release of parental protein was observed. Approx. 40% of the parental protein became degraded and could be washed from the infected cell after 90 min; the rest of the parental protein remained and probably was re-utilized by the host. (+info)
Nonselective coupling of the human mu-opioid receptor to multiple inhibitory G-protein isoforms.
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The human mu-opioid receptor was expressed in Saccharomyces cerevisiae. Binding of [3H]diprenorphine to yeast spheroplasts was specific and saturable (Kd = 1 nm, Bmax = 0.2-1 pmol x mg-1 of membrane proteins). Inhibition of [3H]diprenorphine binding by antagonists and agonists with varying opioid selectivities (mu, delta and kappa) occurred with the same order of potency as in mammalian tissues. Affinities of antagonists were the same with yeast spheroplasts as in reference tissues whereas those of agonists, except etorphine and buprenorphine, were 10-fold to 100-fold lower. Addition of heterotrimeric Gi,o-proteins purified from bovine brain shifted the mu-opioid receptor into a high-affinity state for agonists. Using individually purified Galpha-subunits re-associated with betagamma-dimers, we showed that alphao1, alphao2, alphai1, alphai2 and alphai3 reconstituted high-affinity agonist binding with equal efficiency. This suggests that the structural determinants of the mu-opioid receptor responsible for G-protein coupling are not able to confer a high degree of specificity towards any member of the Gi,o family. The selective effects of opioid observed in specialized tissues upon opioid stimulation may be a result of regulation of G-protein activity by cell-specific factors which should conveniently be analysed using the reconstitution assay described here. (+info)
Dislocation of membrane proteins in FtsH-mediated proteolysis.
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Escherichia coli FtsH degrades several integral membrane proteins, including YccA, having seven transmembrane segments, a cytosolic N-terminus and a periplasmic C-terminus. Evidence indicates that FtsH initiates proteolysis at the N-terminal cytosolic domain. SecY, having 10 transmembrane segments, is also a substrate of FtsH. We studied whether and how the FtsH-catalyzed proteolysis on the cytosolic side continues into the transmembrane and periplasmic regions using chimeric proteins, YccA-(P3)-PhoA-His6-Myc and SecY-(P5)-PhoA, with the alkaline phosphatase (PhoA) mature sequence in a periplasmic domain. The PhoA domain that was present within the fusion protein was rapidly degraded by FtsH when it lacked the DsbA-dependent folding. In contrast, both PhoA itself and the TM9-PhoA region of SecY-(P5)-PhoA were stable when expressed as independent polypeptides. In the presence of DsbA, the FtsH-dependent degradation stopped at a site near to the N-terminus of the PhoA moiety, leaving the PhoA domain (and its C-terminal region) undigested. The efficiency of this degradation stop correlated well with the rapidity of the folding of the PhoA domain. Thus, both transmembrane and periplasmic domains are degraded by the processive proteolysis by FtsH, provided they are not tightly folded. We propose that FtsH dislocates the extracytoplasmic domain of a substrate, probably using its ATPase activity. (+info)
Competition between Sec- and TAT-dependent protein translocation in Escherichia coli.
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Recently, a new protein translocation pathway, the twin-arginine translocation (TAT) pathway, has been identified in both bacteria and chloroplasts. To study the possible competition between the TAT- and the well-characterized Sec translocon-dependent pathways in Escherichia coli, we have fused the TorA TAT-targeting signal peptide to the Sec-dependent inner membrane protein leader peptidase (Lep). We find that the soluble, periplasmic P2 domain from Lep is re-routed by the TorA signal peptide into the TAT pathway. In contrast, the full-length TorA-Lep fusion protein is not re-routed into the TAT pathway, suggesting that Sec-targeting signals in Lep can override TAT-targeting information in the TorA signal peptide. We also show that the TorA signal peptide can be converted into a Sec-targeting signal peptide by increasing the hydrophobicity of its h-region. Thus, beyond the twin-arginine motif, the overall hydrophobicity of the signal peptide plays an important role in TAT versus Sec targeting. This is consistent with statistical data showing that TAT-targeting signal peptides in general have less hydrophobic h-regions than Sec-targeting signal peptides. (+info)
Quinolone accumulation by Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli.
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The accumulation of nalidixic acid and 14 fluoroquinolones over a range of external drug concentrations (10-100 mg/L; c. 25-231 microM) into intact cells of Escherichia coli KL-16, Staphylococcus aureus NCTC 8532, Pseudomonas aeruginosa NCTC 10662 and spheroplasts of E. coli was investigated. The effect of 100 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) upon the concentration of quinolone accumulated by intact cells and spheroplasts of E. coli was also determined. Except for pefloxacin, there was an increase in the concentration of the six quinolones examined accumulated by E. coli, despite a reduction in fluorescence at alkaline pH. For ciprofloxacin the partition coefficient (P(app)) was constant despite an increase in the pH; however, the P(app) for nalidixic acid decreased significantly with an increase in pH. The concentration of nalidixic acid, ciprofloxacin and enrofloxacin accumulated by E. coli and S. aureus increased with an increase in temperature up to 40 degrees C and 50 degrees C, respectively. Above these temperatures the cell viability decreased. With an increase in drug concentration there was, for intact E. coli and 12/15 agents, and for S. aureus and 10/15 agents, a linear increase in the concentration of drug accumulated. However, for P. aeruginosa and 13/15 agents there was apparent saturation of an accumulation pathway. Assuming 100% accumulation into intact cells of E. coli, for 10/14 fluoroquinolones < or = 40% was accumulated by spheroplasts. CCCP increased the concentration of quinolone accumulated but the increase varied with the agent and the bacterial species. The variation in the effect of CCCP upon accumulation of the different quinolones into E. coli could result from chemical interactions or from different affinities of the proposed efflux transporter for each quinolone. Overall, these data suggest that accumulation of most quinolones into E. coli and S. aureus proceeds by simple diffusion, but that P. aeruginosa behaves differently. (+info)
A cell-free assay allows reconstitution of Vps33p-dependent transport to the yeast vacuole/lysosome.
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We report a cell-free system that measures transport-coupled maturation of carboxypeptidase Y (CPY). Yeast spheroplasts are lysed by extrusion through polycarbonate filters. After differential centrifugation, a 125,000-g pellet is enriched for radiolabeled proCPY and is used as "donor" membranes. A 15,000-g pellet, harvested from nonradiolabeled cells and enriched for vacuoles, is used as "acceptor" membranes. When these membranes are incubated together with ATP and cytosolic extracts, approximately 50% of the radiolabeled proCPY is processed to mature CPY. Maturation was inhibited by dilution of donor and acceptor membranes during incubation, showed a 15-min lag period, and was temperature sensitive. Efficient proCPY maturation was possible when donor membranes were from a yeast strain deleted for the PEP4 gene (which encodes the principal CPY processing enzyme, proteinase A) and acceptor membranes from a PEP4 yeast strain, indicating intercompartmental transfer. Cytosol made from a yeast strain deleted for the VPS33 gene was less efficient at driving transport. Moreover, antibodies against Vps33p (a Sec1 homologue) and Vam3p (a Q-SNARE) inhibited transport >90%. Cytosolic extracts from yeast cells overexpressing Vps33p restored transport to antibody-inhibited assays. This cell-free system has allowed the demonstration of reconstituted intercompartmental transport coupled to the function of a VPS gene product. (+info)
The spheroplast lysis assay for yeast in microtiter plate format.
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A yeast lysis assay in the microtiter plate format improved precision and throughput and led to an improved algorithm for estimating lag time. The assay reproducibly revealed differences of 10% or greater in the maximal lysis rate and 50% or greater in the lag time. Clonal differences were determined to be the major source of variation. Microtiter-based assays should be useful for screening for drug susceptibility and for analyzing mutant phenotypes. (+info)