C. van der Does

The bacterial translocase: a dynamic protein channel complex.

The major route of protein translocation in bacteria is the so-called general secretion pathway (Sec-pathway). This route has been extensively studied in Escherichia coli and other bacteria. The movement of preproteins across the cytoplasmic membrane is mediated by a multimeric membrane protein complex called translocase. The core of the translocase consists of a proteinaceous channel formed by an oligomeric assembly of the heterotrimeric membrane protein complex SecYEG and the peripheral adenosine triphosphatase (ATPase) SecA as molecular motor. Many secretory proteins utilize the molecular chaperone SecB for targeting and stabilization of the unfolded state prior to translocation, while most nascent inner membrane proteins are targeted to the translocase by the signal recognition particle and its membrane receptor. Translocation is driven by ATP hydrolysis and the proton motive force. In the last decade, genetic and biochemical studies have provided detailed insights into the mechanism of preprotein translocation. Recent crystallographic studies on SecA, SecB and the SecYEG complex now provide knowledge about the structural features of the translocation process. Here, we will discuss the mechanistic and structural basis of the translocation of proteins across and the integration of membrane proteins into the cytoplasmic membrane.

Non-bilayer Lipids Stimulate the Activity of the Reconstituted Bacterial Protein Translocase*

To determine the phospholipid requirement of the preprotein translocase in vitro, the Escherichia coli SecYEG complex was purified in a delipidated form using the detergent dodecyl maltoside. SecYEG was reconstituted into liposomes composed of defined synthetic phospholipids, and proteoliposomes were analyzed for their preprotein translocation and SecA translocation ATPase activity. The activity strictly required the presence of anionic phospholipids, whereas the non-bilayer lipid phosphatidylethanolamine was found stimulatory. The latter effect could also be induced by dioleoylglycerol, a lipid that adopts a non-bilayer conformation. Phosphatidylethanolamine derivatives that prefer the bilayer state were unable to stimulate translocation. In the absence of SecG, activity was reduced, but the phospholipid requirement was unaltered. Remarkably, non-bilayer lipids were found essential for the activity of the Bacillus subtilis SecYEG complex. Optimal activity required a mixture of anionic and non-bilayer lipids at concentrations that correspond to concentrations found in the natural membrane.

SecA is an intrinsic subunit of the Escherichia coli preprotein translocase and exposes its carboxyl terminus to the periplasm

SecA is the dissociable ATPase subunit of the Escherichia coli preprotein translocase, and cycles in a nucleotide‐modulated manner between the cytosol and the membrane. Overproduction of the integral subunits of the translocase,the SecY, SecE and SecG polypeptides, results in an increased level of membrane‐bound SecA. This fraction of SecA is firmly associated with the membrane as it is resistant to extraction with the chaotropic agent urea, and appears to be anchored by SecYEG rather than by lipids. Topology analysis of this membrane‐associated form of SecA indicates that it exposes a carboxy‐terminal domain to the periplasmic face of the membrane.

The positive inside rule is not determined by the polarity of the Delta psi

The transmembrane orientation of integral membrane proteins from many sources is described by the so-called cispositive or positive-inside rule (Von Heijne, 1986, EMBO J 5: 3021–3027; Von Heijne, 1992, J Mol Biol 225: 487–494; Wallin and Von Heijne, 1998, Prot Sci 7: 1029–1038). The positively charged amino acid residues arginine and lysine are enriched in cytoplasmic domains and extremely rare in extracytoplasmic domains. It has been suggested that the transmembrane electrical potential, Dc, is the main determinant of the topology of membrane proteins (Andersson and Von Heijne, 1994, EMBO J 13: 2267–2272). In most organisms, the Dc is inside negative and outside positive and is supposed to retard the transmembrane passage of positive charges. Here, we report data that contradict such an essential role for Dc in determining the asymmetric distribution of positive charges of membrane proteins. Obligate acidophiles, such as Sulfolobus acidocaldarius, are organisms that thrive at very low environmental pH values (pH 0.5–2.5). These organisms maintain their intracellular pH near neutrality. As a result of the massive transmembrane pH gradient, acidophiles have a reversed Dc, i.e. inside positive instead of negative (Moll and Schäfer, 1988, FEBS Lett 232: 359–363). If the Dc were a major cause of the positive inside rule, an inversed charge distribution is expected for membrane proteins of acidophiles. To test this prediction, we searched databanks for proteins with known topology that also exist in acidophiles. We focused on the homologues of SecY, a subunit of the precursor protein translocase, and on subunit 1 of cytochrome c oxidase. The transmembrane orientations of both proteins are established (Akiyama and Ito, 1989, J Biol Chem 264: 437–442; Tsukihara et al., 1996, Science 272: 1136–1144). Homologues of these proteins were taken from representatives from all three kingdoms (Bacteria, Archaea and Eukarya) and were aligned using CLUSTALW (Thompson et al., 1994, Nucleic Acids Res 22: 4673– 4680). The membrane segments were identified on the basis of hydrophobicity and sequence similarity. Subsequently, the number of charged residues in each hydrophilic loop was scored. Table 1 shows the results for more than nine homologues after assuming the same orientation for all of them (Nin 1 Cin for SecY and Cox1 homologues with 10 and 12 hydrophobic segments respectively). The distribution of arginines and lysines over the hydrophilic loops is the same for all organisms including the acidophiles and agrees with the positive inside rule. In addition, the distribution of the negatively charged residues aspartate and glutamate was similar for all organisms (results not shown). These observations were confirmed for the bitopic SecE homologues from several acidophiles. It is concluded that the positive inside rule is not determined by the polarity of Dc, but is a result of the membrane protein insertion process. In this process anionic lipids, which could anchor the positive charges to the cytosolic leaflet of the membrane Molecular Microbiology (1998) 29(4), 1125–1127

A Single Amino Acid Substitution in SecY Stabilizes the Interaction with SecA

The SecYEG complex constitutes a protein conducting channel across the bacterial cytoplasmic membrane. It binds the peripheral ATPase SecA to form the translocase. When isoleucine 278 in transmembrane segment 7 of the SecY subunit was replaced by a unique cysteine, SecYEG supported an increased preprotein translocation and SecA translocation ATPase activity, and allowed translocation of a preprotein with a defective signal sequence. SecY(I278C)EG binds SecA with a higher affinity than normal SecYEG, in particular in the presence of ATP. The increased translocation activity of SecY(I278C)EG was confirmed in a purified system consisting of SecYEG proteoliposomes, while immunoprecipitation in detergent solution reveal that translocase-preprotein complexes are more stable with SecY(I278C) than with normal SecY. These data imply an important role for SecY transmembrane segment 7 in SecA binding. As improved SecA binding to SecY was also observed with the prlA4 suppressor mutation, it may be a general mechanism underlying signal sequence suppression.

Protein export in bacteria

Publisher Summary This chapter provides an explanation about the protein export in bacteria. The cytoplasmic membrane of bacteria is the site for energy-transducing processes such as respiration, ATP synthesis, solute transport, and flagellar movement. The molecular mechanism of bacterial protein export has been studied in great detail during the last two decades using the powerful genetic and biochemical tools that are available for E. coli. General chaperones such as GroEL and DnaK can substitute for SecB in stabilizing preproteins in a translocation competent state, but they cannot substitute for the specific targeting function of SecB in protein export. The interaction between SecA and the major channel subunit SecY involves multiple regions of both proteins. The actual protein-conducting channel is lined up by four SecYEG complexes. The latter is not essential for translocation, and although it may be functionally homologous, it is not similar to the bacterial SecG. The mechanism underlying thermal sensitivity of protein export is not known, but potential cold-sensitive steps could be insertion of the signal sequence and/or SecA into the membrane, or the oligomerization of the SecYEG complex into a protein-conducting channel. Finally on a concluding note, this chapter summarizes three historical notes as well.