The signal recognition particle-targeting pathway does not necessarily deliver proteins to the sec-translocase in Escherichia coli.

ProW is an Escherichia coli inner membrane protein that consists of a 100-residue-long periplasmic N-terminal tail (N-tail) followed by seven closely spaced transmembrane segments. N-tail translocation presumably proceeds in a C-to-N-terminal direction and represents a poorly understood aspect of membrane protein biogenesis. Here, using an in vivo depletion approach, we show that N-tail translocation in a ProW derivative comprising the N-tail and the first transmembrane segment fused to the globular P2 domain of leader peptidase depends both on the bacterial signal recognition particle (SRP) and the Sec-translocase. Surprisingly, however, a deletion construct with only one transmembrane segment downstream of the N-tail can assemble properly even under severe depletion of SecE, a central component of the Sec-translocase, but not under SRP-depletion conditions. To our knowledge, this is the first demonstration that the SRP-targeting pathway does not necessarily deliver SRP-dependent inner membrane proteins to the Sec-translocase. The data further suggest that N-tail translocation can proceed in the absence of a functional Sec-translocase.     

An artificial transmembrane segment directs SecA, SecB, and electrochemical potential-dependent translocation of a long amino-terminal tail

Many integral membrane proteins contain an amino-terminal segment, often referred to as an N-tail, that is translocated across a membrane. In many cases, translocation of the N-tail is initiated by a cleavable, amino-terminal signal peptide. For N-tail proteins lacking a signal peptide, translocation is initiated by a transmembrane segment that is carboxyl to the translocated segment. The mechanism of membrane translocation of these segments, although poorly understood, has been reported to be independent of the protein secretion machinery. In contrast, here we describe alkaline phosphatase mutants containing artificial transmembrane segments that demonstrate that translocation of a long N-tail across the membrane is dependent upon SecA, SecB, and the electrochemical potential in the absence of a signal peptide. The corresponding mutants containing signal peptides also use the secretion machinery but are less sensitive to inhibition of its components. We present evidence that inhibition of SecA by sodium azide is incomplete even at high concentrations of inhibitor, which suggests why SecA-dependent translocation may not have been detected in other systems. Furthermore, by varying the charge around the transmembrane segment, we find that in the absence of a signal peptide, the orientation of the membrane-bound alkaline phosphatase is dictated by the positive inside rule. However, the presence of a signal peptide is an overriding factor in membrane orientation and renders all mutants in an Nout-Cin orientation.     

Distant downstream sequence determinants can control N-tail translocation during protein insertion into the endoplasmic reticulum membrane

We have studied the membrane insertion of ProW, an Escherichia coli inner membrane protein with seven transmembrane segments and a large periplasmic N-terminal tail, into endoplasmic reticulum (ER)-derived dog pancreas microsomes. Strikingly, significant levels of N-tail translocation is seen only when a minimum of four of the transmembrane segments are present; for constructs with fewer transmembrane segments, the N-tail remains mostly nontranslocated and the majority of the molecules adopt an "inverted" topology where normally nontranslocated parts are translocated and vice versa. N-tail translocation can also be promoted by shortening of the N-tail and by the addition of positively charged residues immediately downstream of the first trasnmembrane segment. We conclude that as many as four consecutive transmembrane segments may be collectively involved in determining membrane protein topology in the ER and that the effects of downstream sequence determinants may vary depending on the size and charge of the N-tail. We also provide evidence to suggest that the ProW N-tail is translocated across the ER membrane in a C-to-N-terminal direction.     

Competition between neighboring topogenic signals during membrane protein insertion into the ER

To better define the mechanism of membrane protein insertion into the membrane of the endoplasmic reticulum, we measured the kinetics of translocation across microsomal membranes of the N-terminal lumenal tail and the lumenal domain following the second transmembrane segment (TM2) in the multispanning mouse protein Cig30. In the wild-type protein, the N-terminal tail translocates across the membrane before the downstream lumenal domain. Addition of positively charged residues to the N-terminal tail dramatically slows down its translocation and allows the downstream lumenal domain to translocate at the same time as or even before the N-tail. When TM2 is deleted, or when the loop between TM1 and TM2 is lengthened, addition of positively charged residues to the N-terminal tail causes TM1 to adopt an orientation with its N-terminal end in the cytoplasm. We suggest that the topology of the TM1-TM2 region of Cig30 depends on a competition between TM1 and TM2 such that the transmembrane segment that inserts first into the ER membrane determines the final topology.     

N-terminal tail export from the mitochondrial matrix. Adherence to the prokaryotic "positive-inside" rule of membrane protein topology.

Export of N-terminal tails of mitochondrial inner membrane proteins from the mitochondrial matrix is a membrane potential-dependent process, mediated by the Oxa1p translocation machinery. The hydrophilic segments of these membrane proteins, which undergo export, display a characteristic charge profile where intermembrane space-localized segments bear a net negative charge, whereas those remaining in the matrix have a net positive one. Using a model protein, preSu9(1-112)-dihydrofolate reductase (DHFR), which undergoes Oxa1p-mediated N-tail export, we demonstrate here that the net charge of N- and C-flanking regions of the transmembrane domain play a critical role in determining the orientation of the insertion process. The N-tail must bear a net negative charge to be exported to the intermembrane space. Furthermore, a net positive charge of the C-terminal region supports this N-tail export event. These data provide experimental evidence that protein export in mitochondria adheres to the "positive-inside" rule, described for sec-independent sorting of membrane proteins in prokaryotes. We propose here that the importance of a charge profile reflects a need for specific protein-protein interactions to occur in the export reaction, presumably at the level of the Oxa1p export machinery.     

Foreign transmembrane peptides replacing the internal signal sequence of transferrin receptor allow its translocation and membrane binding

Each subunit of the human transferrin receptor (TR) dimer is inserted into the ER membrane as a transmembrane polypeptide having its N-terminus in the cytoplasm. The transmembrane segment of the molecule serves both as a signal for chain translocation and as a membrane anchor. To study which structural features of this segment are required for its dual function, we have essentially replaced the transmembrane peptide with the C-terminal membrane-spanning segment of two proteins having a separate N-terminal translocation signal and with an artificial uncharged peptide. In each case the mutant TR molecules are efficiently translocated in vitro. In contrast, substitution of the transmembrane peptide of TR with a hydrophilic peptide results in no detectable translocation activity of the mutant TR. This suggests that the hydrophobic character of the transmembrane peptide of TR, rather than its actual amino acid sequence, is important for chain translocation and membrane binding.     

Arginine 357 of SecY is needed for SecA-dependent initiation of preprotein translocation

The Escherichia coli SecYEG complex forms a transmembrane channel for both protein export and membrane protein insertion. Secretory proteins and large periplasmic domains of membrane proteins require for translocation in addition the SecA ATPase. The conserved arginine 357 of SecY is essential for a yet unidentified step in the SecA catalytic cycle. To further dissect its role, we have analysed the requirement for R357 in membrane protein insertion. Although R357 substitutions abolish post-translational translocation, they allow the translocation of periplasmic domains targeted co-translationally by an N-terminal transmembrane segment. We propose that R357 is essential for the initiation of SecA-dependent translocation only.     

Immobilization of the Plug Domain Inside the SecY Channel Allows Unrestricted Protein Translocation

The SecYEG complex forms a protein-conducting channel in the inner membrane of Escherichia coli to support the translocation of secretory proteins in their unfolded state. The SecY channel is closed at the periplasmic face of the membrane by a small re-entrance loop that connects transmembrane segment 1 with 2b. This helical domain 2a is termed the plug domain. By the introduction of pairs of cysteines and crosslinkers, the plug domain was immobilized inside the channel and connected to transmembrane segment 10. Translocation was inhibited to various degrees depending on the position and crosslinker spacer length. With one of the crosslinked mutants translocation occurred unrestricted. Biochemical characterization of this mutant as well as molecular dynamics simulations suggest that only a limited movement of the plug domain suffices for translocation.     

Mechanism of proton transport by plant plasma membrane proton ATPases

The mechanism of proton translocation by P-type proton ATPases is poorly defined. Asp684 in transmembrane segment M6 of the Arabidopsis thaliana AHA2 plasma membrane P-type proton pump is suggested to act as an essential proton acceptor during proton translocation. Arg655 in transmembrane segment M5 seems to be involved in this proton translocation too, but in contrast to Asp684, is not essential for transport. Asp684 may participate in defining the E₁ proton-binding site, which could possibly exist as a hydronium ion coordination center. A model of proton translocation of AHA2 involving the side chains of amino acids Asp684 and Arg655 is discussed.     

SecY, a multispanning integral membrane protein, contains a potential leader peptidase cleavage site.

SecY is an Escherichia coli integral membrane protein required for efficient translocation of other proteins across the cytoplasmic membrane; it is embedded in this membrane by the 10 transmembrane segments. Among several SecY-alkaline phosphatase (PhoA) fusion proteins that we constructed previously, SecY-PhoA fusion 3-3, in which PhoA is fused to the third periplasmic region of SecY just after the fifth transmembrane segment, was found to be subject to rapid proteolytic processing in vivo. Both the SecY and PhoA products of this cleavage have been identified immunologically. In contrast, cleavage of SecY-PhoA 3-3 was barely observed in a lep mutant with a temperature-sensitive leader peptidase. The full-length fusion protein accumulated in this mutant was cleaved in vitro by the purified leader peptidase. A sequence Ala-202-Ile-Ala located near the proposed interface between transmembrane segment 5 and periplasmic domain 3 of SecY was found to be responsible for the recognition and cleavage by the leader peptidase, since a mutated fusion protein with Phe-Ile-Phe at this position was no longer cleaved even in the wild-type cells. These results indicate that SecY contains a potential leader peptidase cleavage site that undergoes cleavage if the PhoA sequence is attached carboxy terminally. Thus, transmembrane segment 5 of SecY can fulfill both of the two important functions of the signal peptide, translocation and cleavage, although the latter function is cryptic in the normal SecY protein.     

Membrane integration of the second transmembrane segment of band 3 requires a closely apposed preceding signal-anchor sequence

We have investigated the topogenic rules of multispanning membrane proteins using erythrocyte band 3. Here, the fine structural requirements for the correct disposition of its second transmembrane segment (TM2) were assessed. We made fusion proteins where TM1 and the loop sequence preceding TM2 were changed and fused to prolactin. They were expressed in a cell-free system supplemented with rough microsomal membrane, and their topologies on the membrane were assessed by protease sensitivity and N-glycosylation. TM1 was demonstrated to be a signal-anchor sequence that mediates translocation of the downstream portion, and thus TM2 should be responsible to halt the translocation to acquire TM topology. When the loop between TM1 and TM2 was elongated, however, TM2 was readily translocated through the membrane and not integrated. For the membrane integration of TM2, TM2 must be in close proximity to TM1. The TM1 can be replaced with another signal-anchor sequence with a long hydrophobic segment but not with a signal sequence with shorter hydrophobic stretch. The length of the hydrophobic segment affected final topology of TM2. We concluded that the two TM segments work synergistically within the translocon to acquire the correct topology and that the length of the preceding signal sequence is critical for stable transmembrane assembly of TM2. We propose that direct interaction among the TM segments is one of the critical factors for the transmembrane topogenesis of multispanning membrane proteins.     

Translocation of mitochondrially synthesized Cox2 domains from the matrix to the intermembrane space

The N-terminal and C-terminal domains of mitochondrially synthesized cytochrome c oxidase subunit II, Cox2, are translocated through the inner membrane to the intermembrane space (IMS). We investigated the distinct mechanisms of N-tail and C-tail export by analysis of epitope-tagged Cox2 variants encoded in Saccharomyces cerevisiae mitochondrial DNA. Both the N and C termini of a truncated protein lacking the Cox2 C-terminal domain were translocated to the IMS via a pathway dependent upon the conserved translocase Oxa1. The topology of this Cox2 variant, accumulated at steady state, was largely but not completely unaffected in mutants lacking proteins required for export of the C-tail domain, Cox18 and Mss2. C-tail export was blocked by truncation of the last 40 residues from the C-tail domain, indicating that sequence and/or structural features of this domain are required for its translocation. Mss2, a peripheral protein bound to the inner surface of the inner membrane, coimmunoprecipitated with full-length newly synthesized Cox2, whose leader peptide had already been cleaved in the IMS. Our data suggest that the C-tail domain is recognized posttranslationally by a specialized translocation apparatus after the N-tail has been translocated by Oxa1.     

A sugar chain at a specific position in the nascent polypeptide chain induces forward movement during translocation through the translocon

Nascent polypeptide chains synthesized by membrane bound ribosomes are cotranslationally translocated through and integrated into the endoplasmic reticulum translocon. Hydrophobic segments and positive charges on the chain are critical to halt the ongoing translocation. A marginally hydrophobic segment, which cannot be inserted into the membrane by itself, can be a transmembrane segment depending on its downstream positive charges. In certain conditions, positive charges even 60 residues downstream cause the marginally hydrophobic segment to span the membrane by inducing the segment to slide back from the lumen. Here we systematically examined the effect of a core sugar chain on the fate of a marginally hydrophobic segment using a cell-free translation and translocation system. A sugar chain added within 12 residues upstream of the marginally hydrophobic segment prevents the sliding back and promotes forward movement of the polypeptide chain. The sugar chain apparently functions as a ratchet to keep the polypeptide chain in the lumen. We propose that the sugar chain is a third topology determinant of membrane proteins, in addition to a hydrophobic segment and positive charges of the nascent chain.     

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.     

Proprotein convertase PC3 is not a transmembrane protein

Proprotein convertase PC3 (also known as PC1) is an endopeptidase involved in proteolytic processing of peptide hormone precursors in granules of the regulated secretory pathway of endocrine cells. Lacking any extended hydrophobic segments, PC3 was considered to be a secretory protein only peripherally attached to the granule membrane. Recently, evidence has been presented that PC3 is a transmembrane protein with a 115-residue cytoplasmic domain and a membrane-spanning segment containing eight charged amino acids [Arnaoutova, I., et al. (2003) Biochemistry 42, 10445-10455]. Here, we analyzed the membrane topology of PC3 and of a PC3 construct containing a conventional transmembrane segment of 19 leucines. Alkaline extraction was performed to assess membrane integration. Exposure to the cytosol or to the ER lumen was tested by addition of C-terminal tags for phosphorylation or glycosylation, respectively. Protease sensitivity was assayed in permeabilized cells. The results show that the C-terminus of PC3 is translocated across the endoplasmic reticulum membrane. Furthermore, the proposed transmembrane segment of PC3 and a similar one of carboxypeptidase E did not stop polypeptide translocation when inserted into a stop-transfer tester construct. PC3 is thus not a transmembrane protein. These results have implications for the mechanism of granule sorting of PC3 as well as for the topology of PC2 and carboxypeptidase E, which have been reported to span the lipid membrane by homologous charged sequences.     

Disposition of polar and nonpolar residues on outer surfaces of transmembrane helical segments of proteins involved in proton translocation

"Helical wheel" projections of transmembrane helical segments of membrane proteins involved in proton translocation were constructed. The particular proteins studied were the uncF protein subunit of the Escherichia coli proton-ATPase, the uncE protein subunit of the E. coli proton-ATPase, and cytochrome oxidase subunit III. Clear demarcation of polar and nonpolar regions on surfaces of transmembrane helical segments was seen in the uncF protein and in uncE protein helical segment two, but not in uncE protein helical segment one. The transmembrane segment of cytochrome oxidase subunit III which includes the dicyclohexylcarbodiimide (DCCD)-reactive residue was very similar to E. coli uncE protein helical segment two. The DCCD-reactive residue in both was clearly located on a nonpolar surface.     

Stop-transfer efficiency of marginally hydrophobic segments depends on the length of the carboxy-terminal tail

Hydrophobic stop-transfer sequences generally serve to halt the translocation of polypeptide chains across the endoplasmic reticulum membrane and become integrated as transmembrane α-helices. Using engineered glycosylation sites as topology reporters, we show that the length of the nascent chain between a hydrophobic segment and the carboxy terminus of the protein can affect stop-transfer efficiency. We also show that glycosylation sites located close to a protein's C terminus are modified in two distinct kinetic phases, one fast and one slow. Our findings suggest that membrane integration of a hydrophobic segment is not simply a question of thermodynamic equilibrium, but can be influenced by details of the translocation mechanism.     

The transmembrane segment of Tom20 is recognized by Mim1 for docking to the mitochondrial TOM complex

Mitochondria cannot be made de novo. Mitochondrial biogenesis requires that up to 1000 proteins are imported into mitochondria, and the protein import pathway relies on hetero-oligomeric translocase complexes in both the inner and outer mitochondrial membranes. The translocase in the outer membrane, the TOM complex, is composed of a core complex formed from the β-barrel channel Tom40 and additional subunits each with single, α-helical transmembrane segments. How α-helical transmembrane segments might be assembled onto a transmembrane β-barrel in the context of a membrane environment is a question of fundamental importance. The master receptor subunit of the TOM complex, Tom20, recognizes the targeting sequence on incoming mitochondrial precursor proteins, binds these protein ligands, and then transfers them to the core complex for translocation across the outer membrane. Here we show that the transmembrane segment of Tom20 contains critical residues essential for docking the Tom20 receptor into its correct environment within the TOM complex. This crucial docking reaction is catalyzed by the unique assembly factor Mim1/Tom13. Mutations in the transmembrane segment that destabilize Tom20, or deletion of Mim1, prevent Tom20 from functioning as a receptor for protein import into mitochondria.     

Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis

During protein integration into the endoplasmic reticulum, the N-terminal domain preceding the type I signal-anchor sequence is translocated through a translocon. By fusing a streptavidin-binding peptide tag to the N terminus, we created integration intermediates of multispanning membrane proteins. In a cell-free system, N-terminal domain (N-domain) translocation was arrested by streptavidin and resumed by biotin. Even when N-domain translocation was arrested, the second hydrophobic segment mediated translocation of the downstream hydrophilic segment. In one of the defined intermediates, two hydrophilic segments and two hydrophobic segments formed a transmembrane disposition in a productive state. Both of the translocating hydrophilic segments were crosslinked with a translocon subunit, Sec61α. We conclude that two translocating hydrophilic segment in a single membrane protein can span the membrane during multispanning topogenesis flanking the translocon. Furthermore, even after six successive hydrophobic segments entered the translocon, N-domain translocation could be induced to restart from an arrested state. These observations indicate the remarkably flexible nature of the translocon.     

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.