An amino-terminal signal sequence abrogates the intrinsic membrane-targeting information of mitochondrial uncoupling protein

Mitochondrial uncoupling protein, a polytopic integral protein of the inner membrane, is initially made in the cytoplasm as a soluble polypeptide (307 amino acids) lacking a cleavable targeting (signal) peptide. Earlier studies (Liu, X., Bell, A. W., Freeman, K. B., and Shore, G. C. (1988) J. Cell Biol. 107, 503-509) identified internal regions of the molecule that are critical for targeting and membrane insertion. Here, we demonstrate that the ability of uncoupling protein to insert into the inner membrane is abrogated when the molecule is fused behind the matrix-targeting signal of preornithine carbamyltransferase; the hybrid protein was imported across the inner membrane and deposited in the matrix where it was processed. In this context, however, the processed product remained in the matrix and was incapable of inserting into the inner membrane.     

The Structure of Multiple Polypeptide Domains Determines the Signal Recognition Particle Targeting Requirement of Escherichia coli Inner Membrane Proteins

The signal recognition particle (SRP) targeting pathway is required for the efficient insertion of many polytopic inner membrane proteins (IMPs) into the Escherichia coli inner membrane, but in the absence of SRP protein export proceeds normally. To define the properties of IMPs that impose SRP dependence, we analyzed the targeting requirements of bitopic IMPs that are structurally intermediate between exported proteins and polytopic IMPs. We found that disruption of the SRP pathway inhibited the insertion of only a subset of bitopic IMPs. Studies on a model bitopic AcrB-alkaline phosphatase fusion protein (AcrB 265-AP) showed that the SRP requirement for efficient insertion correlated with the presence of a large periplasmic domain (P1). As previously reported, perturbation of the SRP pathway also affected the insertion of a polytopic AcrB-AP fusion. Even exhaustive SRP depletion, however, failed to block the insertion of any AcrB derivative by more than 50%. Taken together, these data suggest that many proteins that are normally targeted by SRP can utilize alternative targeting pathways and that the structure of both hydrophilic and membrane-spanning domains determines the degree to which the biogenesis of a protein is SRP dependent.     

YidC, an assembly site for polytopic Escherichia coli membrane proteins located in immediate proximity to the SecYE translocon and lipids

Like its mitochondrial homolog Oxa1p, the inner membrane protein YidC of Escherichia coli is involved in the integration of membrane proteins. We have analyzed individual insertion steps of the polytopic E. coli membrane protein MtlA targeted as ribosome-nascent chain complexes to inner membrane vesicles. YidC can accommodate at least the first two transmembrane segments of MtlA at the protein lipid interface and retain them even though the length of the nascent chain would amply allow insertion into membrane lipids. An even longer insertion intermediate of MtlA is described that still has the first transmembrane helix bound to YidC while the third contacts SecE and YidC during integration. Our findings suggest that YidC forms a contiguous integration unit with the SecYE translocon and functions as an assembly site for polytopic membrane proteins mediating the formation of helix bundles prior to their release into the membrane lipids.     

The conserved third transmembrane segment of YidC contacts nascent Escherichia coli inner membrane proteins

Escherichia coli YidC is a polytopic inner membrane protein that plays an essential and versatile role in the biogenesis of inner membrane proteins. YidC functions in Sec-dependent membrane insertion but acts also independently as a separate insertase for certain small membrane proteins. We have used a site-specific cross-linking approach to show that the conserved third transmembrane segment of YidC contacts the transmembrane domains of both nascent Sec-dependent and -independent substrates, indicating a generic recognition of insertion intermediates by YidC. Our data suggest that specific residues of the third YidC transmembrane segment alpha-helix is oriented toward the transmembrane domains of nascent inner membrane proteins that, in contrast, appear quite flexibly positioned at this stage in biogenesis.     

The amino-terminal domain of the lamin B receptor is a nuclear envelope targeting signal

The lamin B receptor (LBR) is a polytopic protein of the inner nuclear membrane. It is synthesized without a cleavable amino-terminal signal sequence and composed of a nucleoplasmic amino-terminal domain of 204 amino acids followed by a hydrophobic domain with eight putative transmembrane segments. To identify a nuclear envelope targeting signal, we have examined the cellular localization by immunofluorescence microscopy of chicken LBR, its amino-terminal domain and chimeric proteins transiently expressed in transfected COS-7. Full- length LBR was targeted to the nuclear envelope. The amino-terminal domain, without any transmembrane segments, was transported to the nucleus but excluded from the nucleolus. When the amino-terminal domain of LBR was fused to the amino-terminal side of a transmembrane segment of a type II integral membrane protein of the ER/plasma membrane, the chimeric protein was targeted to the nuclear envelope, likely the inner nuclear membrane. When the amino-terminal domain was deleted from LBR and replaced by alpha-globin, the chimeric protein was retained in the ER. These findings demonstrate that the amino-terminal domain of LBR is targeted to the nucleus after synthesis in the cytoplasm and that this polypeptide can function as a nuclear envelope targeting signal when located at the amino terminus of a type II integral membrane protein synthesized on the ER.     

Dissecting the translocase and integrase functions of the Escherichia coli SecYEG translocon

Recent evidence suggests that in Escherichia coli, SecA/SecB and signal recognition particle (SRP) are constituents of two different pathways targeting secretory and inner membrane proteins to the SecYEG translocon of the plasma membrane. We now show that a secY mutation, which compromises a functional SecY-SecA interaction, does not impair the SRP-mediated integration of polytopic inner membrane proteins. Furthermore, under conditions in which the translocation of secretory proteins is strictly dependent on SecG for assisting SecA, the absence of SecG still allows polytopic membrane proteins to integrate at the wild-type level. These results indicate that SRP-dependent integration and SecA/SecB-mediated translocation do not only represent two independent protein delivery systems, but also remain mechanistically distinct processes even at the level of the membrane where they engage different domains of SecY and different components of the translocon. In addition, the experimental setup used here enabled us to demonstrate that SRP-dependent integration of a multispanning protein into membrane vesicles leads to a biologically active enzyme.     

The “+70 Pause”: Hypothesis of a Translational Control of Membrane Protein Assembly

Sequences of 66 genes encoding bacterial or yeast membrane proteins have been examined for the respective positioning of putative transmembrane domains and translational pauses. The latter were operationally defined as clusters of at least 17 non-preferred codons along the mRNA. The putative transmembrane domains were defined as stretches of at least 17 hydrophobic amino acids in the encoded protein. For yeast non-mitochon drial membrane proteins, it was observed that clusters of non-preferred codons occur more frequently about 56 to 75 codons after a hydrophobic stretch in the encoded protein. About 40 amino acid residues are required to span the large ribosomal subunit. Such clusters were thus predicted to cause a severe slow-down in peptide elongation, just when the hydrophobic stretch fully protrudes from the ribosome. This transient slow-down of the ribosome pace has consequently been named the “+70 pause”. This pause was not observed for mitochondrial or bacterial membrane proteins, which are thought to insert post-translationally in their respective membranes. Because insertion of yeast proteins in the endoplasmic reticulum membrane is generally cotranslational instead, it is possible that the “+70 pause” reflects the coupling of translation, targeting, insertion and folding in this case. The pause may, for instance, give time for productive interaction of the newly synthesized hydrophobic domain with the proper targeting/insertion machineries. Thus, it would favor entrance of the stalled protein domain into the proper pathway.     

Expression of the Cymbidium ringspot virus 33-kilodalton protein in Saccharomyces cerevisiae and molecular dissection of the peroxisomal targeting signal

Open reading frame 1 in the viral genome of Cymbidium ringspot virus encodes a 33-kDa protein (p33), which was previously shown to localize to the peroxisomal membrane in infected and transgenic plant cells. To determine the sequence requirements for the organelle targeting and membrane insertion, the protein was expressed in the yeast Saccharomyces cerevisiae in native form (33K) or fused to the green fluorescent protein (33KGFP). Cell organelles were identified by immunolabeling of marker proteins. In addition, peroxisomes were identified by simultaneous expression of the red fluorescent protein DsRed containing a peroxisomal targeting signal and mitochondria by using the dye MitoTracker. Fluorescence microscopy showed the 33KGFP fusion protein concentrated in a few large bodies colocalizing with peroxisomes. These bodies were shown by electron microscopy to be composed by aggregates of peroxisomes, a few mitochondria and endoplasmic reticulum (ER) strands. In immunoelectron microscopy, antibodies to p33 labeled the peroxisomal clumps. Biochemical analysis suggested that p33 is anchored to the peroxisomal membrane through a segment of ca. 7 kDa, which corresponds to the sequence comprising two hydrophobic transmembrane domains and a hydrophilic interconnecting loop. Analysis of deletion mutants confirmed these domains as essential components of the p33 peroxisomal targeting signal, together with a cluster of three basic amino acids (KRR). In yeast mutants lacking peroxisomes p33 was detected in the ER. The possible involvement of the ER as an intermediate step for the integration of p33 into the peroxisomal membrane is discussed.     

Signal peptidase can cleave inside a polytopic membrane protein

The signal peptides of most proteins targeted to the endoplasmic reticulum are specifically cleaved by signal peptidase. Although potential cleavage sites occur frequently in polytopic proteins after membrane-spanning segments, processing is restricted to the first hydrophobic domain, suggesting that signal peptidase might not have access to subsequently translocated, internal domains. To test this hypothesis, we replaced the third transmembrane segment of an artificial threefold membrane-spanning protein by a sequence which is normally an amino-terminal signal. Upon in vitro translation and insertion into microsomes, efficient cleavage at this sequence was observed, thus demonstrating the ability of signal peptidase to cleave within polytopic membrane proteins.     

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.     

Sec-translocase mediated membrane protein biogenesis

Alpha-helical transmembrane proteins in bacteria are localized within the plasma membrane. The membrane assembly of these proteins requires protein devices for insertion into the lipid bilayer. In E. coli, membrane proteins require the SRP pathway components Ffh, 4.5S RNA and FtsY for membrane targeting and the SecYEGDF translocase and, in some cases, SecA, for translocation of hydrophilic domains. In addition, YidC, a recently discovered membrane protein, mediates the membrane integration and folding of hydrophobic domains of membrane proteins. In this review, we will describe the current status of the protein targeting and membrane integration pathways.     

The retinal rod Na(+)/Ca(2+),K(+) exchanger contains a noncleaved signal sequence required for translocation of the N terminus

The retinal rod Na(+)/Ca(2+),K(+) exchanger (RodX) is a polytopic membrane protein found in photoreceptor outer segments where it is the principal extruder of Ca(2+) ions during light adaptation. We have examined the role of the N-terminal 65 amino acids in targeting, translocation, and integration of the RodX using an in vitro translation/translocation system. cDNAs encoding human RodX and bovine RodX through the first transmembrane domain were correctly targeted and integrated into microsomal membranes; deletion of the N-terminal 65 amino acids (aa) resulted in a translation product that was not targeted or integrated. Deletion of the first 65 aa had no effect on membrane targeting of full-length RodX, but the N-terminal hydrophilic domain no longer translocated. Chimeric constructs encoding the first 65 aa of bovine RodX fused to globin were translocated across microsomal membranes, demonstrating that the sequence could function heterologously. Studies of fresh bovine retinal extracts demonstrated that the first 65 aa are present in the native protein. These data demonstrate that the first 65 aa of RodX constitute an uncleaved signal sequence required for the efficient membrane targeting and proper membrane integration of RodX.     

Tim23p Contains Separate and Distinct Signals for Targeting to Mitochondria and Insertion into the Inner Membrane

The Tim23 protein is an essential inner membrane (IM) component of the yeast mitochondrial protein import pathway. Tim23p does not carry an amino-terminal presequence; therefore, the targeting information resides within the mature protein. Tim23p is anchored in the IM via four transmembrane segments and has two positively charged loops facing the matrix. To identify the import signal for Tim23p, we have constructed several altered versions of the Tim23 protein and examined their function and import in yeast cells, as well as their import into isolated mitochondria. We replaced the positively charged amino acids in one or both loops with alanine residues and found that the positive charges are not required for import into mitochondria, but at least one positively charged loop is required for insertion into the IM. Furthermore, we find that the signal to target Tim23p to mitochondria is carried in at least two of the hydrophobic transmembrane segments. Our results suggest that Tim23p contains separate import signals: hydrophobic segments for targeting Tim23p to mitochondria, and positively charged loops for insertion into the IM. We therefore propose that Tim23p is imported into mitochondria in at least two distinct steps.     

Membrane topology of the murine fatty acid transport protein 1

The murine fatty acid transport protein (FATP1) was identified in an expression cloning screen for proteins that facilitate transport of fatty acids across the plasma membranes of mammalian cells. Hydropathy analysis of this protein suggests a model in which FATP1 has multiple membrane-spanning domains. To test this model, we inserted a hemagglutinin epitope tag at the amino terminus or a FLAG tag at the carboxyl terminus of the FATP1 cDNA and expressed these constructs in NIH 3T3 cells. Both tagged constructs produce proteins of the expected molecular masses and are functional in fatty acid import assays. Indirect immunofluorescence studies with selective permeabilization conditions and protease protection studies of sealed membrane vesicles from cells expressing epitope-tagged FATP1 were performed. These experiments show that the extreme amino terminus of tagged FATP1 is oriented toward the extracellular space, whereas the carboxyl terminus faces the cytosol. Additionally, enhanced green fluorescent protein fusion constructs containing predicted membrane-associated or soluble portions of FATP1 were expressed in Cos7 cells and analyzed by immunofluorescence and subcellular fractionation. These experiments demonstrate that amino acids 1-51, 52-100, and 101-190 contain signals for integral association with the membrane, whereas residues 258-313 and 314-475 are only peripherally membrane-associated. Amino acid residues 191-257 and 476-646 do not direct membrane association and likely face the cytosol. Taken together, these data support a model of FATP1 as a polytopic membrane protein with at least one transmembrane and multiple membrane-associated domains. This study provides the first experimental evidence for topology of a member of the family of plasma membrane fatty acid transport proteins.     

Export of beta-lactamase is independent of the signal recognition particle

In Escherichia coli, three different types of proteins engage the SecY translocon of the inner bacterial membrane for translocation or insertion: 1) polytopic membrane proteins that prior to their insertion into the membrane are targeted to the translocon using the bacterial signal recognition particle (SRP) and its receptor; 2) secretory proteins that are targeted to and translocated across the SecY translocon in a SecA- and SecB-dependent reaction; and 3) membrane proteins with large periplasmic domains, requiring SRP for targeting and SecA for the translocation of the periplasmic moiety. In addition to its role as a targeting device for membrane proteins, a function of the bacterial SRP in the export of SecB-independent secretory proteins has also been postulated. In particular, beta-lactamase, a hydrolytic enzyme responsible for cleavage of the beta-lactam ring containing antibiotics, is considered to be recognized and targeted by SRP. To examine the role of the SRP pathway in beta-lactamase targeting and export, we performed a detailed in vitro analysis. Chemical cross-linking and membrane binding assays did not reveal any significant interaction between SRP and beta-lactamase nascent chains. More importantly, membrane vesicles prepared from mutants lacking a functional SRP pathway did block the integration of SRP-dependent membrane proteins but supported the export of beta-lactamase in the same way as that of the SRP-independent protein OmpA. These data demonstrate that in contrast to previous results, the bacterial SRP is not involved in the export of beta-lactamase and further suggest that secretory proteins of Gram-negative bacteria in general are not substrates of SRP.     

Topogenesis of peroxisomal membrane protein requires a short, positively charged intervening-loop sequence and flanking hydrophobic segments. study using human membrane protein PMP34

Human 34-kDa peroxisomal membrane protein (PMP34) consisting of 307 amino acids was previously identified as an ortholog of, or a similar protein (with 27% identity) to the, 423-amino acid-long PMP47 of the yeast Candida boidinii. We investigated membrane topogenesis of PMP34 with six putative transmembrane segments, as a model peroxisomal membrane protein. PMP34 was characterized as an integral membrane protein of peroxisomes. Transmembrane topology of PMP34 was determined by differential permeabilization and immunofluorescent staining of HeLa cells ectopically expressing PMP34 as well as of Chinese hamster ovary-K1 expressing epitope-tagged PMP34. As opposed to PMP47, PMP34 was found to expose its N- and C-terminal parts to the cytosol. Various deletion variants of PMP34 and their fusion proteins with green fluorescent protein were expressed in Chinese hamster ovary-K1 and were verified with respect to intracellular localization. The loop region between transmembrane segments 4 and 5 was required for the peroxisome-targeting activity, in which Ala substitution for basic residues abrogated the activity. Three hydrophobic transmembrane segments linked in a flanking region of the basic loop were essential for integration of PMP34 to peroxisome membranes. Therefore, it is evident that the intervening basic loop plus three transmembrane segments of PMP34 function as a peroxisomal targeting and topogenic signal.     

Topological and functional studies on HlyB of Escherichia coli

Summary The topology of HlyB, a protein located in the inner membrane of Escherichia coli and involved in the secretion of -haemolysin (HlyA), was determined by the generation of HlyB-PhoA and HlyB-LacZ fusion proteins. The data obtained by this biochemical method together with computer predictions suggest that HlyB is inserted in the cytoplasmic membrane by six stable hydrophobic, -helical transmembrane segments. These segments extend from amino acid positions 158 to 432 of HlyB. The cytoplasmic loops between these transmembrane segments are relatively large and carry an excess of positively charged amino acids, while the periplasmic loops are rather small. In addition to these six transmembrane segments, two additional regions in the 78 N-terminal amino acids of HlyB appear to be also inserted in the cytoplasmic membrane. However, the association of these two segments with the cytoplasmic membrane seems to be less tight, since active PhoA and LacZ fusions were obtained by insertion into the same positions of these segments. A LacZ-HlyAs fusion protein carrying, at the C-terminus of LacZ, the 60-amino acid signal sequence of HlyA was not secreted in the presence of HlyB/HlyD. However, transport of this fusion protein into the cytoplasmic membrane appeared to be initiated, as suggested by the tight association of this protein with the inner membrane. A similar close association of LacZ-HlyA_s with the inner membrane was also observed in the presence of HlyB alone but not in its absence. These data suggest that HlyB recognizes the HlyA signal sequence and initiates the transport of HlyA into the membrane.     

Identification of an outer segment targeting signal in the COOH terminus of rhodopsin using transgenic Xenopus laevis

Mislocalization of the photopigment rhodopsin may be involved in the pathology of certain inherited retinal degenerative diseases. Here, we have elucidated rhodopsin's targeting signal which is responsible for its polarized distribution to the rod outer segment (ROS). Various green fluorescent protein (GFP)/rhodopsin COOH-terminal fusion proteins were expressed specifically in the major red rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The fusion proteins were targeted to membranes via lipid modifications (palmitoylation and myristoylation) as opposed to membrane spanning domains. Membrane association was found to be necessary but not sufficient for efficient ROS localization. A GFP fusion protein containing only the cytoplasmic COOH-terminal 44 amino acids of Xenopus rhodopsin localized exclusively to ROS membranes. Chimeras between rhodopsin and alpha adrenergic receptor COOH-terminal sequences further refined rhodopsin's ROS localization signal to its distal eight amino acids. Mutations/deletions of this region resulted in partial delocalization of the fusion proteins to rod inner segment (RIS) membranes. The targeting and transport of endogenous wild-type rhodopsin was unaffected by the presence of mislocalized GFP fusion proteins.     

Different transmembrane domains associate with distinct endoplasmic reticulum components during membrane integration of a polytopic protein

We have been studying the insertion of the seven transmembrane domain (TM) protein opsin to gain insights into how the multiple TMs of polytopic proteins are integrated at the endoplasmic reticulum (ER). We find that the ER components associated with the first and second TMs of the nascent opsin polypeptide chain are clearly distinct. The first TM (TM1) is adjacent to the alpha and beta subunits of the Sec61 complex, and a novel component, a protein associated with the ER translocon of 10 kDa (PAT-10). The most striking characteristic of PAT-10 is that it remains adjacent to TM1 throughout the biogenesis and membrane integration of the full-length opsin polypeptide. TM2 is also found to be adjacent to Sec61alpha and Sec61beta during its membrane integration. However, TM2 does not form any adducts with PAT-10; rather, a transient association with the TRAM protein is observed. We show that the association of PAT-10 with opsin TM1 does not require the N-glycosylation of the nascent chain and occurs irrespective of the amino acid sequence and transmembrane topology of TM1. We conclude that the precise makeup of the ER membrane insertion site can be distinct for the different transmembrane domains of a polytopic protein. We find that the environment of a particular TM can be influenced by both the "stage" of nascent chain biosynthesis reached, and the TM's relative location within the polypeptide.     

An amphiphilic region in the cytoplasmic domain of KdpD is recognized by the signal recognition particle and targeted to the Escherichia coli membrane

The sensor protein KdpD of Escherichia coli is composed of a large N-terminal hydrophilic region (aa 1–400), four transmembrane regions (aa 401–498) and a large hydrophilic region (aa 499–894) at the C-terminus. KdpD requires the signal recognition particle (SRP) for its targeting to the membrane. Deletions within KdpD show that the first 50 residues are required for SRP-driven membrane insertion. A fusion protein of the green fluorescent protein (GFP) with KdpD is found localized at the membrane only when SRP is present. The membrane targeting of GFP was not observed when the first 50 KdpD residues were deleted. A truncated mutant of KdpD containing only the first 25 amino acids fused to GFP lost its ability to specifically interact with SRP, whereas a specific interaction between SRP and the first 48 amino acids of KdpD fused to GFP was confirmed by pull-down experiments. Conclusively, a small amphiphilic region of 27 residues within the amino-terminal domain of KdpD (aa 22–48) is recognized by SRP and targets the protein to the membrane. This shows that membrane proteins with a large N-terminal region in the cytoplasm can be membrane-targeted early on to allow co-translational membrane insertion of their distant transmembrane regions.