YidC, the Escherichia coli homologue of mitochondrial Oxa1p, is a component of the Sec translocase

In Escherichia coli, both secretory and inner membrane proteins initially are targeted to the core SecYEG inner membrane translocase. Previous work has also identified the peripherally associated SecA protein as well as the SecD, SecF and YajC inner membrane proteins as components of the translocase. Here, we use a cross-linking approach to show that hydrophilic portions of a co-translationally targeted inner membrane protein (FtsQ) are close to SecA and SecY, suggesting that insertion takes place at the SecA/Y interface. The hydrophobic FtsQ signal anchor sequence contacts both lipids and a novel 60 kDa translocase-associated component that we identify as YidC. YidC is homologous to Saccharomyces cerevisiae Oxa1p, which has been shown to function in a novel export pathway at the mitochondrial inner membrane. We propose that YidC is involved in the insertion of hydrophobic sequences into the lipid bilayer after initial recognition by the SecAYEG translocase.     

Dislocation by the m-AAA Protease Increases the Threshold Hydrophobicity for Retention of Transmembrane Helices in the Inner Membrane of Yeast Mitochondria

Sorting of mitochondrial inner membrane proteins is a complex process in which translocons and proteases function in a concerted way. Many inner membrane proteins insert into the membrane via the TIM23 translocon, and some are then further acted upon by the mitochondrial m-AAA protease, a molecular motor capable of dislocating proteins from the inner membrane. This raises the possibility that the threshold hydrophobicity for the retention of transmembrane segments in the inner membrane is different depending on whether they belong to membrane proteins that are m-AAA protease substrates or not. Here, using model transmembrane segments engineered into m-AAA protease-dependent proteins, we show that the threshold hydrophobicity for membrane retention measured in yeast cells in the absence of a functional m-AAA protease is markedly lower than that measured in its presence. Whether a given hydrophobic segment in a mitochondrial inner membrane protein will ultimately form a transmembrane helix may therefore depend on whether or not it will be exposed to the pulling force exerted by the m-AAA protease during biogenesis.     

Proline residues of transmembrane domains determine the sorting of inner membrane proteins in mitochondria

Most inner membrane proteins of mitochondria are synthesized in the cytosol and reach the inner membrane using one of two alternative sorting pathways. On the stop transfer route, proteins are arrested during import at the level of the inner membrane. The conservative sorting pathway involves translocation through the inner membrane and insertion from the matrix. It is unclear how the translocase of the inner membrane 23 protein translocation machinery differentiates between the two classes of proteins. Here we show that proline residues in hydrophobic stretches strongly disfavor the translocation arrest of transmembrane domains (TMDs) and favor the transfer of preproteins to the matrix. We propose that proline residues, together with the hydrophobicity of the TMD and the presence of charged residues COOH-terminally flanking the TMD, are determinants of the intramitochondrial sorting of inner membrane proteins.     

MotX and MotY,specific components of the sodium-driven flagellar motor,colocalize to the outer membrane in Vibrio alginolyticus

Rotation of the sodium-driven polar flagella of Vibrio alginolyticus requires four motor proteins: PomA, PomB, MotX and MotY. MotX and MotY, which are unique components of the sodium-driven motor of Vibrio, have been believed to be localized in the inner (cytoplasmic) membrane via their N-terminal hydrophobic segments. Here we show that MotX and MotY colocalize to the outer membrane. Both proteins, when expressed together, were detected in the outer membrane fraction separated by sucrose density gradient centrifugation. As mature MotX and MotY proteins do not have N-terminal hydrophobic segments, the N-termini of the primary translation products must have signal sequences that are removed upon translocation across the inner membrane. Moreover, MotX and MotY require each other for efficient localization to the outer membrane. Based on these lines of evidence, we propose that MotX and MotY form a complex in the outer membrane. This is the first case that describes motor proteins function in the outer membrane for flagellar rotation.     

The Escherichia coli SRP and SecB targeting pathways converge at the translocon.

Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins. SecB targets the pre-protein to SecA that mediates pre-protein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.     

Mitochondria use different mechanisms for transport of multispanning membrane proteins through the intermembrane space

The mitochondrial inner membrane contains numerous multispanning integral proteins. The precursors of these hydrophobic proteins are synthesized in the cytosol and therefore have to cross the mitochondrial outer membrane and intermembrane space to reach the inner membrane. While the import pathways of noncleavable multispanning proteins, such as the metabolite carriers, have been characterized in detail by the generation of translocation intermediates, little is known about the mechanism by which cleavable preproteins of multispanning proteins, such as Oxa1, are transferred from the outer membrane to the inner membrane. We have identified a translocation intermediate of the Oxa1 preprotein in the translocase of the outer membrane (TOM) and found that there are differences from the import mechanisms of carrier proteins. The intermembrane space domain of the receptor Tom22 supports the stabilization of the Oxa1 intermediate. Transfer of the Oxa1 preprotein to the inner membrane is not affected by inactivation of the soluble TIM complexes. Both the inner membrane potential and matrix heat shock protein 70 are essential to release the preprotein from the TOM complex, suggesting a close functional cooperation of the TOM complex and the presequence translocase of the inner membrane. We conclude that mitochondria employ different mechanisms for translocation of multispanning proteins across the aqueous intermembrane space.     

A subset of bacterial inner membrane proteins integrated by the twin-arginine translocase

A group of bacterial exported proteins are synthesized with N-terminal signal peptides containing a SRRxFLK 'twin-arginine' amino acid motif. Proteins bearing twin-arginine signal peptides are targeted post-translationally to the twin-arginine translocation (Tat) system which transports folded substrates across the inner membrane. In Escherichia coli, most integral inner membrane proteins are assembled by a co-translational process directed by SRP/FtsY, the SecYEG translocase, and YidC. In this work we define a novel class of integral membrane proteins assembled by a Tat-dependent mechanism. We show that at least five E. coli Tat substrate proteins contain hydrophobic C-terminal transmembrane helices (or 'C-tails'). Fusions between the identified transmembrane C-tails and the exclusively Tat-dependent reporter proteins TorA and SufI render the resultant chimeras membrane-bound. Export-linked signal peptide processing and membrane integration of the chimeras is shown to be both Tat-dependent and YidC-independent. It is proposed that the mechanism of membrane integration of proteins by the Tat system is fundamentally distinct from that employed for other bacterial inner membrane proteins.     

Charged flanking residues control the efficiency of membrane insertion of the first transmembrane segment in yeast mitochondrial Mgm1p

Mgm1p is a nuclearly encoded GTPase important for mitochondrial fusion. Long and short isoforms of the protein are generated in a unique "alternative topogenesis" process in which the most N-terminal of two hydrophobic segments in the protein is inserted into the inner mitochondrial membrane in about half of the molecules and translocated across the inner membrane in the other half. In the latter population, the second hydrophobic segment is cleaved by the inner membrane protease Pcp1p, generating the short isoform. Here, we show that charged residues in the regions flanking the first segment critically affect the ratio between the two isoforms, providing new insight into the importance of charged residues in the insertion of proteins into the mitochondrial inner membrane.     

The N-terminal hydrophobic region of the mature phosphate translocator is sufficient for targeting to the chloroplast inner envelope membrane.

To locate the sequence required for directing the phosphate translocator to the chloroplast inner envelope membrane, a series of chimeric proteins constituting parts of the phosphate translocator and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, which is normally located in the stroma, has been produced. Reciprocal exchanges of the presequences and mature sequences of the phosphate translocator and the small subunit indicated that the phosphate translocator presequence contains stromal targeting information and that the mature protein is responsible for inner envelope membrane targeting. Chimeric proteins containing the N-terminal 46 amino acid residues of the phosphate translocator were directed to the inner envelope membrane. Subdivision of this region into its composite hydrophilic and hydrophobic regions showed that the hydrophobic region alone, which consists of amino acid residues 24 to 45, was able to direct the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase to the inner envelope membrane.     

Sequence of gene malG in E. coli K12: homologies between integral membrane components from binding protein-dependent transport systems.

The MalG protein is needed for the transport of maltose in Escherichia coli K12. We present the sequence of gene malG. The deduced amino acid sequence corresponds to a protein of 296 amino acid residues (mol. wt. = 32 188 daltons). This protein is largely hydrophobic (hydrophobic index = 0.83) and is thus presumably an integral inner membrane protein which could span the membrane through six hydrophobic segments. We provide direct evidence from fusion proteins for the translation frame and we also identified the in vitro made MalG protein. We have found a sequence which is highly conserved between MalG and MalF, the other integral inner membrane protein of the maltose transport system. This conserved sequence is also present in all known integral membrane proteins of binding protein-dependent transport systems, always at the same distance (approximately 90 residues) from their COOH terminus. We discuss briefly this finding.     

Interaction of mammalian mitochondrial ribosomes with the inner membrane

All of the products of mitochondrial protein biosynthesis in animals are hydrophobic proteins that are localized in the inner membrane. Hence, it is possible that the synthesis of these proteins could occur on ribosomes associated with the inner membrane. To examine this possibility, inner membrane and matrix fractions of bovine mitochondria were examined for the presence of ribosomes using probes for the rRNAs. Between 40 and 50% of the ribosomes were found to fractionate with the inner membrane. About half of the ribosomes associated with the inner membrane could be released by high salt treatment, indicating that they interact with the membrane largely through electrostatic forces. No release of the ribosome was observed upon treatment with puromycin, suggesting that the association observed is not due to insertion of a nascent polypeptide chain into the membrane. A fraction of the ribosomes remained with residual portions of the membranes that cannot be solubilized in the presence of Triton X-100. These ribosomes may be associated with large oligomeric complexes in the membrane.     

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.     

The essential virulence protein VirB8 localizes to the inner membrane of Agrobacterium tumefaciens.

Agrobacterium tumefaciens genetically transforms plant cells by transferring a specific DNA fragment from the bacterium through several biological membranes to the plant nucleus where the DNA is integrated. This complex DNA transport process likely involves membrane-localized proteins in both the plant and the bacterium. The 11 hydrophobic or membrane-localized proteins of the virB operon are excellent candidates to have a role in DNA export from agrobacteria. Here, we show by TnphoA mutagenesis and immunogold electron microscopy that one of the VirB proteins, VirB8, is located at the inner membrane. The observation that a virB8::TnphoA fusion restores export of alkaline phosphatase to the periplasm suggests that VirB8 spans the inner membrane. Immunogold labeling of VirB8 was detected on the inner membrane of vir-induced A. tumefaciens by transmission electron microscopy. Compared with that of the controls, VirB8 labeling was significantly greater on the inner membrane than on the other cell compartments. These results confirm the inner membrane localization of VirB8 and strengthen the hypothesis that VirB proteins help form a transfer DNA export channel or gate.     

Entropic tension in crowded membranes

Unlike their model membrane counterparts, biological membranes are richly decorated with a heterogeneous assembly of membrane proteins. These proteins are so tightly packed that their excluded area interactions can alter the free energy landscape controlling the conformational transitions suffered by such proteins. For membrane channels, this effect can alter the critical membrane tension at which they undergo a transition from a closed to an open state, and therefore influence protein function in vivo. Despite their obvious importance, crowding phenomena in membranes are much less well studied than in the cytoplasm. Using statistical mechanics results for hard disk liquids, we show that crowding induces an entropic tension in the membrane, which influences transitions that alter the projected area and circumference of a membrane protein. As a specific case study in this effect, we consider the impact of crowding on the gating properties of bacterial mechanosensitive membrane channels, which are thought to confer osmoprotection when these cells are subjected to osmotic shock. We find that crowding can alter the gating energies by more than [Formula: see text] in physiological conditions, a substantial fraction of the total gating energies in some cases. Given the ubiquity of membrane crowding, the nonspecific nature of excluded volume interactions, and the fact that the function of many membrane proteins involve significant conformational changes, this specific case study highlights a general aspect in the function of membrane proteins.     

Lipid fluidity and membrane protein dynamics

Membrane fluidity plays an important role in cellular functions. Membrane proteins are mobile in the lipid fluid environment; lateral diffusion of membrane proteins is slower than expected by theory, due to both the effect of protein crowding in the membrane and to constraints from the aqueous matrix. A major aspect of diffusion is in macromolecular associations: reduction of dimensionality for membrane diffusion facilitates collisional encounters, as those concerned with receptor-mediated signal transduction and with electron transfer chains. In mitochondrial electron transfer, diffusional control is prevented by the excess of collisional encounters between fast-diffusing ubiquinone and the respiratory complexes. Another aspect of dynamics of membrane proteins is their conformational flexibility. Lipids may induce the optimal conformation for catalytic activity. Breaks in Arrhenius plots of membrane-bound enzymes may be related to lipid fluidity: the break could occur when a limiting viscosity is reached for catalytic activity. Viscosity can affect protein conformational changes by inhibiting thermal fluctuations to the inner core of the protein molecule.     

Ribosome binding to the Oxa1 complex facilitates co-translational protein insertion in mitochondria

The Oxa1 translocase of the mitochondrial inner membrane facilitates the insertion of both mitochondrially and nuclear-encoded proteins from the matrix into the inner membrane. Most mitochondrially encoded proteins are hydrophobic membrane proteins which are integrated into the lipid bilayer during their synthesis on mitochondrial ribosomes. The molecular mechanism of this co-translational insertion process is unknown. Here we show that the matrix-exposed C-terminus of Oxa1 forms an alpha-helical domain that has the ability to bind to mitochondrial ribosomes. Deletion of this Oxa1 domain strongly diminished the efficiency of membrane insertion of subunit 2 of cytochrome oxidase, a mitochondrially encoded substrate of the Oxa1 translocase. This suggests that co-translational membrane insertion of mitochondrial translation products is facilitated by a physical interaction of translation complexes with the membrane-bound translocase.     

Differential extraction of hydrophobic proteins from chloroplast envelope membranes: a subcellular-specific proteomic approach to identify rare intrinsic membrane proteins

Identification of rare hydrophobic membrane proteins is a major biological problem that is limited by the specific biochemical approaches required to extract these proteins from membranes and purify them. This is especially true for membranes, such as plastid envelope membranes, that have a high lipid content, present a wide variety of specific functions and therefore contain a large number of unique, but minor, proteins. We have optimized a procedure, based on the differential solubilization of membrane proteins in chloroform/methanol mixtures, to extract and concentrate the most hydrophobic proteins from chloroplast envelope membrane preparations, while more hydrophilic proteins were excluded. In addition to previously characterized chloroplast envelope proteins, such as the phosphate/triose phosphate translocator, we have identified new proteins that were shown to contain putative transmembrane α-helices. Moreover, using different chloroform/methanol mixtures, we have obtained differential solubilization of envelope proteins as a function of their hydrophobicity. All the proteins identified were genuine chloroplast envelope proteins, most of them being localized within the inner membrane. Our procedure enables direct mapping (by classical SDS-PAGE) and identification of hydrophobic membrane proteins, whatever their isoelectric point was, that are minor components of specific subcellular compartments. Thus, it complements other techniques that give access to peripheral membrane proteins. If applied to various cell membranes, it is anticipated that it can expedite the identification of hydrophobic proteins involved in transport systems for ions or organic solutes, or it may act as signal receptors or to control metabolic processes and vesicle trafficking.     

多维蛋白鉴别技术分析天蓝色链霉菌膜内侧蛋白质组

链霉菌是一类具有重要工业价值和复杂调控机制的微生物,天蓝色链霉菌是这个属的模式菌。已报道天蓝色链霉菌的蛋白组学的研究多采用二维聚丙烯酰胺凝胶电泳与基质辅助激光解吸电离飞行时间质谱相结合的方法,但由于膜蛋白疏水性较强,天然丰度较低,此法得到的膜蛋白很少。用蛋白酶K保护/高pH蛋白酶K法制备链霉菌膜内侧蛋白组样品,并用多维蛋白鉴别技术进行分析,得到154个可能的膜内侧蛋白（包括膜内在蛋白和膜外周蛋白）,其中含跨膜区的膜内在蛋白44个,含3个以上跨膜区的膜内在蛋白有23个。此外,还鉴定了一批膜内侧蛋白的亲水性肽段及其在膜上的拓扑位置。该结果有助于对天蓝色链霉菌的膜蛋白进行拓扑学分类和功能研究。     

Dropping Out and Other Fates of Transmembrane Segments Inserted by the SecA ATPase

Type II single-span membrane proteins, such as CadC or RodZ, lacking a signal sequence and having a far-downstream hydrophobic segment, require the SecA secretion motor for insertion into the inner membrane of Escherichia coli. Using two chimeric single-span proteins containing a designed hydrophobic segment H, we have determined the requirements for SecA-mediated secretion, the molecular distinction between TM domains and signal peptides, and the propensity for hydrophobic H-segments to remain embedded within the bilayer after targeting. By means of engineered H-segments and a strategically placed SPase I cleavage site, we determined how targeting and stability of the chimeric proteins are affected by the length and hydrophobicity of the H-segment. Very hydrophobic segments (e.g., 16 Leu) are stably incorporated into the inner membrane, resulting in a C-terminal anchored membrane protein, while a 24L construct was not targeted to the membrane by SecA and remained in the cytoplasm. However, a construct carrying preMalE at the N-terminus led to SecA targeting to SecYEG via the native signal sequence and stable insertion of the downstream 24L H-segment. We show that the RseP intramembrane protease degrades weakly stable H-segments and is a useful tool for investigating the borderline between stable and unstable TM segments. Using RseP⁻ cells, we find that moderately hydrophobic sequences (e.g., 5Leu + 11Ala) are targeted to SecYEG by SecA and inserted, but subsequently drop out of the membrane into the cytoplasm. Therefore, the free energy of transfer from translocon to bilayer is different from the transfer free energy from membrane to water.     

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.