A conserved function of YidC in the biogenesis of respiratory chain complexes.

The Escherichia coli inner membrane protein (IMP) YidC is involved in the membrane integration of IMPs both in concert with and independently from the Sec translocase. YidC seems to be dispensable for the assembly of Sec-dependent IMPs, and so far it has been shown to be essential only for the proper Sec-independent integration of some phage coat proteins. Here, we studied the physiological consequences of YidC depletion in an effort to understand the essential function of YidC. The loss of YidC rapidly and specifically induced the Psp stress response, which is accompanied by a reduction of the proton-motive force. This reduction is due to defects in the functional assembly of cytochrome o oxidase and the F(1)F(o) ATPase complex, which is reminiscent of the effects of mutations in the yidC homologue OXA1 in the yeast mitochondrial inner membrane. The integration of CyoA (subunit II of the cytochrome o oxidase) and F(o)c (membrane subunit of the F(1)F(o) ATPase) appeared exceptionally sensitive to depletion of YidC, suggesting that these IMPs are natural substrates of a membrane integration and assembly pathway in which YidC plays an exclusive or at least a pivotal role.

SecDFyajC is not required for the maintenance of the proton motive force.

SecDFyajC of Escherichia coli is required for efficient export of proteins in vivo. However, the functional role of SecDFyajC in protein translocation is unclear. We evaluated the postulated function of SecDFyajC in the maintenance of the proton motive force. As previously reported, inner membrane vesicles (IMVs) lacking SecDFyajC are defective in the generation of a stable proton motive force when energized with succinate. This phenomenon is, however, not observed when NADH is used as an electron donor. Moreover, the proton motive force generated in SecDFyajC-depleted vesicles stimulated translocation to the same extent as seen with IMVs containing SecDFyajC. Further analysis demonstrates that the reduced proton motive force with succinate in IMVs lacking SecDFyajC is due to a lower amount of the enzyme succinate dehydrogenase. The expression of this enzyme complex is repressed by growth on glucose media, the condition used to deplete SecDFyajC. These results demonstrate that SecDFyajC is not required for proton motive force-driven protein translocation.

Reconstitution of Sec-dependent membrane protein insertion: nascent FtsQ interacts with YidC in a SecYEG-dependent manner.

The inner membrane protein YidC is associated with the preprotein translocase of Escherichia coli and contacts transmembrane segments of nascent inner membrane proteins during membrane insertion. YidC was purified to homogeneity and co-reconstituted with the SecYEG complex. YidC had no effect on the SecA/SecYEG-mediated translocation of the secretory protein proOmpA; however, using a crosslinking approach, the transmembrane segment of nascent FtsQ was found to gain access to YidC via SecY. These data indicate the functional reconstitution of the initial stages of YidC-dependent membrane protein insertion via the SecYEG complex.

Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy.

Secretins, a superfamily of multimeric outer membrane proteins, mediate the transport of large macromolecules across the outer membrane of Gram-negative bacteria. Limited proteolysis of secretin PulD from the Klebsiella oxytoca pullulanase secretion pathway showed that it consists of an N-terminal domain and a protease-resistant C-terminal domain that remains multimeric after proteolysis. The stable C-terminal domain starts just before the region in PulD that is highly conserved in the secretin superfamily and apparently lacks the region at the C-terminal end to which the secretin-specific pilot protein PulS binds. Electron microscopy showed that the stable fragment produced by proteolysis is composed of two stacked rings that encircle a central channel and that it lacks the peripheral radial spokes that are seen in the native complex. Moreover, the electron microscopic images suggest that the N-terminal domain folds back into the large cavity of the channel that is formed by the C-terminal domain of the native complex, thereby occluding the channel, consistent with previous electrophysiological studies showing that the channel is normally closed.

Secretin PulD: association with pilot PulS, structure, and ion-conducting channel formation.

The outer membrane protein PulD (secretin) of Klebsiella oxytoca is required for transport of pullulanase across this membrane. We have purified a multimeric PulD complex from an Escherichia coli strain expressing all the proteins involved in pullulanase secretion. The outer membrane-anchored lipoprotein PulS was found to copurify with PulD. The molar ratio of the two proteins is close to 1:1, and the size of the complex is approximately 1 MDa. Scanning transmission electron and cryo-electron microscopy analyses showed that the purified complex is a cylindrical structure having a central cavity of approximately 7.6 nm and peripheral radial spokes. Fusion of proteoliposomes containing the purified complex with a planar lipid bilayer resulted in the appearance of small, voltage-activated, ion-conducting channels. We conclude that the central cavity seen in the electron microscope is part of a large gated channel and propose that the observed current fluctuations correspond to voltage-induced, relatively minor displacements of domains in the purified complex rather than to a complete opening of the secretin channel.

Folding of a bacterial outer membrane protein during passage through the periplasm.

The transport of bacterial outer membrane proteins to their destination might be either a one-step process via the contact zones between the inner and outer membrane or a two-step process, implicating a periplasmic intermediate that inserts into the membrane. Furthermore, folding might precede insertion or vice versa. To address these questions, we have made use of the known 3D-structure of the trimeric porin PhoE of Escherichia coli to engineer intramolecular disulfide bridges into this protein at positions that are not exposed to the periplasm once the protein is correctly assembled. The mutations did not interfere with the biogenesis of the protein, and disulfide bond formation appeared to be dependent on the periplasmic enzyme DsbA, which catalyzes disulfide bond formation in the periplasm. This proves that the protein passes through the periplasm on its way to the outer membrane. Furthermore, since the disulfide bonds create elements of tertiary structure within the mutant proteins, it appears that these proteins are at least partially folded before they insert into the outer membrane.

prlA suppressors in Escherichia coli relieve the proton electrochemical gradient dependency of translocation of wild-type precursors.

The SecY protein of Escherichia coli is an integral membrane component of the protein export apparatus. Suppressor mutations in the secY gene (prlA alleles) have been isolated that restore the secretion of precursor proteins with defective signal sequences. These mutations have never been shown to affect the translocation of wild-type precursor proteins. Here, we report that prlA suppressor mutations relieve the proton-motive force (pmf) dependency of the translocation of wild-type precursors, both in vivo and in vitro. Furthermore, the proton-motive force dependency of the translocation of a precursor with a stably folded domain in the mature region was suppressed by prlA mutations in vitro. These data show that prlA mutations cause a general relaxation of the export apparatus rather than a specific change that results in bypassing of the recognition of the signal sequence. In addition, these results are indicative for a mechanism in which the proton-motive force stimulates translocation by altering the conformation of the translocon.

Delta mu H+ dependency of in vitro protein translocation into Escherichia coli inner-membrane vesicles varies with the signal-sequence core-region composition.

Signal sequences frequently contain alpha-helix-destabilizing amino acids in the hydrophobic core. Nuclear magnetic resonance studies on the conformation of signal sequences in membrane mimetic environments revealed that these residues cause a break in the alpha-helix. In the precursor of the Escherichia coli outer membrane protein PhoE (pre-PhoE), a glycine residue at position -10 (Gly -10) is thought to be responsible for the break in the alpha-helix. We investigated the role of this glycine residue in the translocation process by employing site-directed mutagenesis. SDS-PAGE analysis showed drastic variations in the electrophoretic mobilities of the mutant precursor proteins, suggesting an important role of the glycine residue in determining the conformation of the signal sequence. In vivo, no drastic differences in the translocation kinetics were observed as compared with wild-type PhoE, except when a charged residue (Arg) was substituted for Gly -10. However, the in vitro translocation of all mutant proteins into inverted inner-membrane vesicles was affected. Two classes of precursors could be distinguished. Translocation of one class of mutant proteins (Ala, Cys and Leu for Gly -10) was almost independent of the presence of a delta mu H+, whereas translocation of the other class of precursors (wild type or Ser) was strongly decreased in the absence of the delta mu H+. Apparently, the delta mu H+ dependency of in vitro protein translocation varies with the signal-sequence core-region composition. Furthermore, a proline residue at position -10 resulted in a signal sequence that did not prevent the folding of the precursor in an in vitro trimerization assay.

Mode of insertion of the signal sequence of a bacterial precursor protein into phospholipid bilayers as revealed by cysteine-based site-directed spectroscopy.

The interactions between a bacterial precursor protein and phospholipids in bilayer-based model membrane systems is addressed in this study. The precursor-lipid interactions were assessed from the side of the lipid phase by fluorescence and electron spin resonance spectroscopy, using the precursor of the Escherichia coli outer membrane protein PhoE. The role of the signal sequence, as part of the precursor, in this interaction was investigated by using cysteine-based site-directed spectroscopy. For this purpose, purified cysteine-containing mutants of prePhoE, which were made by site-directed mutagenesis of the signal sequence part and of the mature part, and defined lipids were used. The location of the fluorescently labeled cysteine residues was established by resonance energy transfer and quenching experiments and those of the corresponding spin-labeled cysteine residues by paramagnetic relaxation enhancement. It was demonstrated that precursor-phospholipid interactions exist in model membrane systems and also that these interactions were dependent on the presence of anionic phospholipids and resulted in a deep insertion of (parts of) the precursor into the lipid bilayer. Furthermore, the results with the cysteine mutations in the signal sequence of the precursor indicate that both termini of the signal sequence are located near or at the membrane surface, with only the fluorescence of the labeled cysteines in the signal sequence part being protected against aqueous quenchers. The results demonstrate that, when part of the intact precursor, the signal sequence experiences similar lipid-protein interactions as do isolated signal peptides. They also indicate that the signal sequence inserts entirely as a looped structure into the membrane. In addition, the data also indicate that the mature part of the precursor has an affinity for the membrane.

The C terminus of SecA is involved in both lipid binding and SecB binding.

Using C-terminal deletion mutations in secA, we localized the previously proposed (Breukink, E., Keller, R.C. A., and de Kruijff, B. (1993), FEBS Lett. 331, 19-24) second lipid binding site on SecA. Since removal of these residues completely abolished the property of SecA to cause aggregation of negatively charged phosphatidyl-glycerol vesicles, we conclude that the C-terminal 70 amino acid residues of SecA are involved in lipid-binding. The C-terminal 70 amino acid residues of SecA are important for efficient in vitro translocation of the SecB-dependent precursor of PhoE across inverted inner membrane vesicles. Moreover, in vivo studies showed that this region is essential for growth. SecB and a SecB-precursor complex were shown to inhibit the SecA-mediated lipid vesicle aggregation, suggesting that the overall acidic SecB protein binds at or near the second lipid binding site on SecA. This together with the observation that the SecA mutant protein lacking the C-terminal 70 residues had a strongly reduced ability to mediate binding of SecB-precursor complexes to inverted inner membrane vesicles demonstrates that the C terminus of SecA is also involved in SecB binding.

Requirement for conformational flexibility in the signal sequence of precursor protein.

According to the "unlooping" model (de Vrije, T., Batenburg, A. M., Killian, J. A., and de Kruijff, B. (1990) Mol. Microbiol. 4, 143-150), proposed to explain how signal sequences serve to target proteins into the secretory pathway, the initial interaction of the signal sequence with the membrane in a helix-turn-helix conformation (spanning half of the bilayer) plays an important role in the initiation of the translocation reaction. To test this model we have introduced 2 cysteines (at positions -5 and -19) in the signal sequence of the Escherichia coli outer membrane protein PhoE. The mutations did not influence the translocation of precursor PhoE in vivo or in vitro. The 2 cysteines were oxidized to form a disulfide bridge. In vitro translocation of the looped precursor into inner membrane vesicles was disturbed. The looped precursor competed with translocation of wild type precursor PhoE, and looped precursor that was first bound to inner membrane vesicles could be translocated after the addition of dithiothreitol. Apparently, the mutant precursor with a disulfide bridge in the signal sequence is arrested as a very early intermediate in the translocation process. All of these results are consistent with the proposed unlooping model and show that, besides the primary structure characteristics of a signal sequence, conformational flexibility is needed to initiate the translocation reaction.

Regulation of glycolytic enzymes and the Crabtree effect in galactose-limited continuous cultures of Saccharomyces cerevisiae.

In order to determine whether the changes in the activities and mRNA levels of enzymes involved in intermediary carbon metabolism previously observed in glucose-limited continuous cultures (Sierkstra et al., 1992a) were glucose specific, we have analysed their regulation in a galactose-limited continuous culture of Saccharomyces cerevisiae. The Vmax of the galactose uptake system was shown to be dilution rate (D) dependent, comparable with the high-affinity glucose uptake. The maximum uptake was observed at D 0.2 h-1 (0.25 mmol min-1 per g) and the minimum uptake (0.1 mmol min-1 per g) at D 0.05 h-1 and 0.3 h-1. The aerobic fermentation of galactose occurred at D 0.275-0.3 h-1 which is identical to the results obtained in glucose-limited continuous cultures of this strain. Because galactose is not a repressing carbon source, this demonstrates that the Crabtree effect is not mediated by, or in any way related to glucose repression. Moreover, invertase and hexokinase I mRNA levels (both subject to glucose repression at the transcriptional level) were present when the yeast produced ethanol in galactose- and glucose-limited continuous cultures. In glucose-limited continuous cultures a decrease in alcohol dehydrogenase (I and II) mRNA levels and activity and phosphoglucomutase activity was observed with increasing dilution rates. In addition, at D 0.3 h-1, when the yeast produced ethanol, glucose-6-phosphate dehydrogenase and pyruvate decarboxylase were induced and a decrease in respiration was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of glucose repression in Saccharomyces cerevisiae by pulsing glucose to a galactose-limited continuous culture.

In this study, glucose repression in Saccharomyces cerevisiae was analysed under defined physiological conditions, at both the molecular and physiological levels, by pulsing glucose to a galactose-limited continuous culture. During this pulse of glucose, the galactose feed was kept constant. Directly after the glucose pulse, carbon dioxide production increased while oxygen consumption remained constant, demonstrating that the surplus of glucose had been consumed by means of fermentation. The direct accumulation of galactose in the medium after the glucose pulse indicated that the consumption of galactose had been stopped instantaneously. Galactose uptake experiments revealed that the galactose transporter was still present but apparently was incapable of galactose uptake, which could be due to inhibition of the galactose transporter by glucose. The total concentration of cAMP increased from 5 nmol g-1 at t = 0 to 25 nmol g-1 at t = 1.5 min. After 2 min the concentration of cAMP gradually decreased again to the normal level. Within 2 min after the addition of glucose, the transcription of the GAL genes and SUC2 was inhibited. In addition, the transcription of the HXK1 gene, encoding hexokinase isoenzyme 1, was also inhibited, which demonstrates that the HXK1 gene is regulated at the transcriptional level comparable with invertase.