A dual function for SecA in the assembly of single spanning membrane proteins in Escherichia coli

The assembly of bacterial membrane proteins with large periplasmic loops is an intrinsically complex process because the SecY translocon has to coordinate the signal recognition particle-dependent targeting and integration of transmembrane domains with the SecA-dependent translocation of the periplasmic loop. The current model suggests that the ATP hydrolysis by SecA is required only if periplasmic loops larger than 30 amino acids have to be translocated. In agreement with this model, our data demonstrate that the signal recognition particle- and SecA-dependent multiple spanning membrane protein YidC becomes SecA-independent if the large periplasmic loop connecting transmembrane domains 1 and 2 is reduced to less than 30 amino acids. Strikingly, however, we were unable to render single spanning membrane proteins SecA-independent by reducing the length of their periplasmic loops. For these proteins, the complete assembly was always SecA-dependent even if the periplasmic loop was reduced to 13 amino acids. If, however, the 13-amino acid-long periplasmic loop was fused to a downstream transmembrane domain, SecA was no longer required for complete translocation. Although these data support the current model on the SecA dependence of multiple spanning membrane proteins, they indicate a novel function of SecA for the assembly of single spanning membrane proteins. This could suggest that single and multiple spanning membrane proteins are processed differently by the bacterial SecY translocon.     

Topology of the retinal cone NCKX2 Na/Ca-K exchanger

The Na/Ca-K exchanger (NCKX) is a polytopic membrane protein that plays a critical role in Ca(2+) homeostasis in retinal rod and cone photoreceptors. The NCKX1 isoform is found in rods, while the NCKX2 isoform is found in cones, in retinal ganglion cells, and in various parts of the brain. The topology of the Na/Ca-K exchanger is thought to consist of two large hydrophilic loops and two sets of transmembrane spanning segments (TMs). The first large hydrophilic loop is located extracellularly at the N-terminus; the other is cytoplasmic and separates the two sets of TMs. The TMs consist of either five and five membrane spanning helices or five and six membrane spanning helices, depending upon the predictive algorithm used. Little specific information is yet available on the orientation of the various membrane spanning helices and the localization of the short loops connecting these helices. In this study, we have determined which of the connecting loops are exposed to the extracellular milieu using two different methods: accessibility of substituted cysteine residues and insertion of N-glycosylation sites. The two methods resulted in a consistent NCKX topology in which the two sets of TMs each contain five membrane spanning helices. Our new model places what was previously membrane spanning helix six in the cytoplasm, which places the C-terminus on the extracellular surface. Surprisingly, this NCKX topology model is different from the current NCX topology model with respect to the C-terminal three membrane helices.     

Membrane topology of the xenobiotic-exporting subunit, MexB, of the MexA,B-OprM extrusion pump in Pseudomonas aeruginosa

The MexA,B-OprM efflux pump assembly of Pseudomonas aeruginosa consists of two inner membrane proteins and one outer membrane protein. The cytoplasmic membrane protein, MexB, appears to function as the xenobiotic-exporting subunit, whereas the MexA and OprM proteins are supposed to function as the membrane fusion protein and the outer membrane channel protein, respectively. Computer-aided hydropathy analyses of MexB predicted the presence of up to 17 potential transmembrane segments. To verify the prediction, we analyzed the membrane topology of MexB using the alkaline phosphatase gene fusion method. We obtained the following unique characteristics. MexB bears 12 membrane spanning segments leaving both the amino and carboxyl termini in the cytoplasmic side of the inner membrane. Both the first and fourth periplasmic loops had very long hydrophilic domains containing 311 and 314 amino acid residues, respectively. This fact suggests that these loops may interact with other pump subunits, such as the membrane fusion protein MexA and the outer membrane protein OprM. Alignment of the amino- and the carboxyl-terminal halves of MexB showed a 30% homology and transmembrane segments 1, 2, 3, 4, 5, and 6 could be overlaid with the segments 7, 8, 9, 10, 11, and 12, respectively. This result suggested that the MexB has a 2-fold repeat that strengthen the experimentally determined topology model. This paper reports the structure of the pump subunit, MexB, of the MexA,B-OprM efflux pump assembly. This is the first time to verify the topology of the resistant-nodulation-division efflux pump protein.     

Genome-wide analysis of integral membrane proteins from eubacterial,archaean, and eukaryotic organisms.

We have carried out detailed statistical analyses of integral membrane proteins of the helix-bundle class from eubacterial, archaean, and eukaryotic organisms for which genome-wide sequence data are available. Twenty to 30% of all ORFs are predicted to encode membrane proteins, with the larger genomes containing a higher fraction than the smaller ones. Although there is a general tendency that proteins with a smaller number of transmembrane segments are more prevalent than those with many, uni-cellular organisms appear to prefer proteins with 6 and 12 transmembrane segments, whereas Caenorhabditis elegans and Homo sapiens have a slight preference for proteins with seven transmembrane segments. In all organisms, there is a tendency that membrane proteins either have many transmembrane segments with short connecting loops or few transmembrane segments with large extra-membraneous domains. Membrane proteins from all organisms studied, except possibly the archaeon Methanococcus jannaschii, follow the so-called "positive-inside" rule; i.e., they tend to have a higher frequency of positively charged residues in cytoplasmic than in extra-cytoplasmic segments.     

Comparative analysis of amino acid distributions in integral membrane proteins from 107 genomes

We have performed a comparative analysis of amino acid distributions in predicted integral membrane proteins from a total of 107 genomes. A procedure for identification of membrane spanning helices was optimized on a homology-reduced data set of 170 multi-spanning membrane proteins with experimentally determined topologies. The optimized method was then used for extraction of highly reliable partial topologies from all predicted membrane proteins in each genome, and the average biases in amino acid distributions between loops on opposite sides of the membrane were calculated. The results strongly support the notion that a biased distribution of Lys and Arg residues between cytoplasmic and extra-cytoplasmic segments (the positive-inside rule) is present in most if not all organisms.     

Membrane topology of the Na(+)/citrate transporter CitS of Klebsiella pneumoniae by insertion mutagenesis

The sodium ion dependent citrate transporter of Klebsiella pneumoniae (CitS) is a member of the bacterial 2-hydroxycarboxylate transporter family. Membrane topology models of the protein, largely based on reporter molecule fusions to C-terminally truncated CitS molecules, indicate that the protein traverses the membrane 11 times with the NH(2)-terminus in the cytoplasm and the COOH-terminus in the periplasm. Furthermore, the structure is characterized by unusual long loops in the COOH-terminal half of the protein: one hydrophobic segment between transmembrane segments V and VI in the periplasm and three long loops connecting transmembrane segments VI and VII, VIII and IX and X and XI in the cytoplasm. The 10 kDa biotin acceptor domain and six consecutive His residues (His-tag) were inserted at different positions in the four long loops and the effect on transport activity and protein stability was analyzed. Six out of seven insertion mutants were stably expressed and three of these had retained significant transport activity. The sidedness of the tags in the mutants that tolerated the insertion was determined by proteolysis experiments. The results support the 11 transmembrane segment model that was based upon truncated CitS proteins.     

Topogenic signals in integral membrane proteins

Integral membrane proteins are characterized by long apolar segments that cross the lipid bilayer. Polar domains flanking these apolar segments have a more balanced amino acid composition, typical for soluble proteins. We show that the apolar segments from three different kinds of membrane-assembly signals do not differ significantly in amino acid content, but that the inside/outside location of the polar domains correlates strongly with their content of arginyl and lysyl residues, not only for bacterial inner-membrane proteins, but also for eukaryotic.proteins from the endoplasmic reticulum, the plasma membrane, the inner mitochondrial membrane, and the chloroplast thylakoid membrane. A positive-inside rule thus seems to apply universally to all integral membrane proteins, with apolar regions targeting for membrane integration and charged residues providing the topological information.     

Anionic phospholipids are determinants of membrane protein topology.

The orientation of many membrane proteins is determined by the asymmetric distribution of positively charged amino acid residues in cytoplasmic and translocated loops. The positive-inside rule states that loops with large amounts of these residues tend to have cytoplasmic locations. Orientations of constructs derived from the inner membrane protein leader peptidase from Escherichia coli were found to depend on the anionic phospholipid content of the membrane. Lowering the contents of anionic phospholipids facilitated membrane passage of positively charged loops. On the other hand, elevated contents of acidic phospholipids in the membrane rendered translocation more sensitive to positively charged residues. The results demonstrate that anionic lipids are determinants of membrane protein topology and suggest that interactions between negatively charged phospholipids and positively charged amino acid residues contribute to the orientation of membrane proteins.     

Leader peptidase

The Escherichia coli leader peptidase has been vital for unravelling problems in membrane assembly and protein export. The role of this essential peptidase is to remove amino-terminal leader peptides from exported proteins after they have crossed the plasma membrane. Strikingly, almost all periplasmic proteins, many outer membrane proteins, and a few inner membrane proteins are made with cleavable leader peptides that are removed by this peptidase. This enzyme of 323 amino acid residues spans the membrane twice, with its large carboxyl-terminal domain protruding into the periplasm. Recent discoveries show that its membrane orientation is controlled by positively charged residues that border (on the cytosolic side) the transmembrane segments. Cleavable pre-proteins must have small residues at -1 and a small or aliphatic residue at -3 (with respect to the cleavage site). Leader peptidase does not require a histidine or cysteine amino acid for catalysis. Interestingly, serine 90 and aspartic acid 153 are essential for catalysis and are also conserved in a mitochondrial leader peptidase, which is 30.7% homologous with the bacterial enzyme over a 101-residue stretch.     

The mitochondrial intermembrane loop region of rat carnitine palmitoyltransferase 1A is a major determinant of its malonyl-CoA sensitivity

Carnitine palmitoyltransferase (CPT) 1A adopts a polytopic conformation within the mitochondrial outer membrane, having both the N- and C-terminal segments on the cytosolic aspect of the membrane and a loop region connecting the two transmembrane (TM) segments protruding into the inter membrane space. In this study we demonstrate that the loop exerts major effects on the sensitivity of the enzyme to its inhibitor, malonyl-CoA. Insertion of a 16-residue spacer between the C-terminal part of the loop sequence (i.e. between residues 100 and 101) and TM2 (which is predicted to start at residue 102) increased the sensitivity to malonyl-CoA inhibition of the resultant mutant protein by more than 10-fold. By contrast, the same insertion made between TM1 and the loop had no effects on the kinetic properties of the enzyme, indicating that effects on the catalytic C-terminal segment were specifically induced by loop-TM2 interactions. Enhanced sensitivity was also observed in all mutants in which the native TM2-loop pairing was disrupted either by making chimeras in which the loops and TM2 segments of CPT 1A and CPT 1B were exchanged or by deleting successive 9-residue segments from the loop sequence. The data suggest that the sequence spanning the loop-TM2 boundary determines the disposition of this TM in the membrane so as to alter the conformation of the C-terminal segment and thus affect its interaction with malonyl-CoA.     

Insertion of bitopic membrane proteins into the inner membrane of mitochondria involves an export step from the matrix

The mitochondrial inner membrane contains a large number of polytopic proteins that are derived from prokaryotic ancestors of mitochondria. Little is known about the intramitochondrial sorting of these proteins. We chose two proteins of known topology as examples to study the pathway of insertion into the inner membrane; Mrs2 and Yta10 are bitopic proteins that expose negatively charged loops of different complexity into the intermembrane space. Here we show that both Mrs2 and Yta10 transiently accumulate as sorting intermediates in the matrix before they integrate into the inner membrane. The sorting pathway of both proteins can be separated into two sequential reactions: (i) import into the matrix and (ii) insertion from the matrix into the inner membrane. The latter process was found to depend on the membrane potential and, in this respect, is similar to the insertion of membrane proteins in bacteria. A comparison of the charge distribution of intermembrane space loops in a variety of mitochondrial inner membrane proteins suggests that this mode of "conservative sorting" might be the typical insertion route for polytopic inner membrane proteins that originated from bacterial ancestors.     

Positively charged residues are important determinants of membrane protein topology

Membrane proteins are found in a variety of conformations, with each protein spanning the membrane a set number of times and adopting a particular orientation. Positively charged residues, often located near the boundaries of transmembrane segments, appear to be involved in specifying the topology of membrane proteins.     

Tryptophan supports interaction of transmembrane helices

Interactions of transmembrane helices play an important role in folding and oligomerization of integral membrane proteins. The interfacial residues of these helices frequently correspond to heptad repeat motifs. In order to uncover novel mechanisms underlying these interactions, we randomised a heptad repeat pattern with a complete set of amino acids. Those sequences that were capable of high-affinity self-interaction upon integration into bacterial inner membranes were selected by means of the POSSYCCAT system. A comparison between selected and non-selected sequences reveals that high-affinity sequences were strongly enriched in tryptophan residues that accumulated at specific positions of the heptad motif. Mutation of Trp in selected clones significantly reduced self-interaction of the transmembrane segments without affecting their efficiency of membrane integration. Conversely, grafting Trp onto artificial transmembrane segments strongly enhanced their interaction. We conclude that tryptophan supports interaction of transmembrane segments.     

The role of the Tim8p-Tim13p complex in a conserved import pathway for mitochondrial polytopic inner membrane proteins

Tim23p is imported via the TIM (translocase of inner membrane)22 pathway for mitochondrial inner membrane proteins. In contrast to precursors with an NH2-terminal targeting presequence that are imported in a linear NH2-terminal manner, we show that Tim23p crosses the outer membrane as a loop before inserting into the inner membrane. The Tim8p-Tim13p complex facilitates translocation across the intermembrane space by binding to the membrane spanning domains as shown by Tim23p peptide scans with the purified Tim8p-Tim13p complex and crosslinking studies with Tim23p fusion constructs. The interaction between Tim23p and the Tim8p-Tim13p complex is not dependent on zinc, and the purified Tim8p-Tim13p complex does not coordinate zinc in the conserved twin CX3C motif. Instead, the cysteine residues seemingly form intramolecular disulfide linkages. Given that proteins of the mitochondrial carrier family also pass through the TOM (translocase of outer membrane) complex as a loop, our study suggests that this translocation mechanism may be conserved. Thus, polytopic inner membrane proteins, which lack an NH2-terminal targeting sequence, pass through the TOM complex as a loop followed by binding of the small Tim proteins to the hydrophobic membrane spanning domains.     

Defining the regions of Escherichia coli YidC that contribute to activity

The YidC/Oxa1/Alb3 family of proteins catalyzes membrane protein insertion in bacteria, mitochondria, and chloroplasts. In this study, we investigated which regions of the bacterial YidC protein are important for its function in membrane protein biogenesis. In Escherichia coli, YidC spans the membrane six times, with a large 319-residue periplasmic domain following the first transmembrane domain. We found that this large periplasmic domain is not required for YidC function and that the residues in the exposed hydrophilic loops or C-terminal tail are not critical for YidC activity. Rather, the five C-terminal transmembrane segments that contain the three consensus sequences in the YidC/Oxa1/Alb3 family are important for its function. However, by systematically replacing all the residues in transmembrane segment (TM) 2, TM3, and TM6 with serine and by swapping TM4 and TM5 with unrelated transmembrane segments, we show that the precise sequence of these transmembrane regions is not essential for in vivo YidC activity. Single serine mutations in TM2, TM3, and TM6 impaired the membrane insertion of the Sec-independent procoat-leader peptidase protein. We propose that the five C-terminal transmembrane segments of YidC function as a platform for the translocating substrate protein to support its insertion into the membrane.     

Sequences of the Escherichia coli BtuB protein essential for its insertion and function in the outer membrane.

The Escherichia coli btuB gene encodes the outer membrane transporter for vitamin B12, the E colicins, colicin A, and bacteriophage BF23. Several series of mutant forms of BtuB resulting from the insertion of dipeptide sequences and from overlapping in-frame deletions and duplications were constructed. Strains expressing the variant genes in single and multiple copy numbers were analyzed for BtuB function, for the level of BtuB polypeptide in the outer membrane, and for changes in the outer membrane permeability barrier. Most dipeptide insertions had normal transport function and assembly in the membrane. Only 2 of the 27 deletions spanning residues 5 and 514 possessed transport function, and most of the remainder were not stably inserted in the membrane. Most duplications (19 of 21) retained transport function and were inserted in the outer membrane, although some were subject to proteolysis. Even long duplications containing as many as 340 repeated amino-terminal residues retained function, suggesting considerable plasticity in the sequence requirements for membrane insertion of BtuB. Expression of many deletion and duplication proteins conferred increased susceptibility to structurally unrelated inhibitors that are normally excluded by the outer membrane. These results could be consistent with the mutational disruption of extracellular loops or transmembrane segments of BtuB that constitute a gated channel, but the finding that alterations throughout the length of BtuB affect membrane permeability properties suggests that the altered proteins might perturb the outer membrane structure itself.     

Major coat proteins of bacteriophage Pf3 and M13 as model systems for Sec-independent protein transport

Abstract: The membrane insertion of bacteriophage coat proteins occurs independent of the Sec-translocase of Escherichia coli . Detailed study of the Pf3 and M13 coat proteins has elucidated two fundamental mechanisms of how proteins invade the membrane, most likely by direct interaction with the lipid bilayer. The Sec-independent translocation of amino-terminal regions across the inner membrane is limited to a short length and a small number of charged residues. Protein regions that contain several charged residues are efficiently translocated across the membrane when these regions are flanked by two adjacent hydrophobic segments interacting synergistically. The relevance of these findings for the membrane insertion mechanism of multispanning membrane proteins is discussed.     

Receptor tyrosine kinases.

A major process through which environmental information is transmitted into cells is via activation of protein tyrosine kinases. Receptor tyrosine kinases contain extracellular ligand recognition, single membrane spanning, and cytoplasmic protein tyrosine kinase domains. The cytoplasmic kinase core is flanked by regulatory segments, which in some family members are also inserted into the core kinase domain. Ligand binding initiates receptor signaling from the cell surface. Activated receptors autophosphorylate to remove alternate substrate/inhibitory constraints and to provide loci for assembly of proteins that contain SRC homology regions. Information is transmitted and diffused by tyrosine phosphorylation of the assembled proteins and of cellular substrates that include protein kinases with specificity for serine/threonine residues. Signaling, which is strictly ligand-dependent, is attenuated by down-regulation of receptors and by feed-back inhibitory loops that involve receptor phosphorylation by cellular kinases. The tyrosine kinase receptors are essential for normal growth, development, and reparative processes. Mutations that remove normal regulatory constraints on the approximately 290 amino acid kinase core of these large proteins result in constitutive function and cell transformation.     

Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation

Enveloped viruses enter cells via a membrane fusion reaction driven by conformational changes of specific viral envelope proteins. We report here the structure of the ectodomain of the tick-borne encephalitis virus envelope glycoprotein, E, a prototypical class II fusion protein, in its trimeric low-pH-induced conformation. We show that, in the conformational transition, the three domains of the neutral-pH form are maintained but their relative orientation is altered. Similar to the postfusion class I proteins, the subunits rearrange such that the fusion peptide loops cluster at one end of an elongated molecule and the C-terminal segments, connecting to the viral transmembrane region, run along the sides of the trimer pointing toward the fusion peptide loops. Comparison with the low-pH-induced form of the alphavirus class II fusion protein reveals striking differences at the end of the molecule bearing the fusion peptides, suggesting an important conformational effect of the missing membrane connecting segment.