TRAP assists membrane protein topogenesis at the mammalian ER membrane

Membrane protein insertion and topogenesis generally occur at the Sec61 translocon in the endoplasmic reticulum membrane. During this process, membrane spanning segments may adopt two distinct orientations with either their N- or C-terminus translocated into the ER lumen. While different topogenic determinants in membrane proteins, such as flanking charges, polypeptide folding, and hydrophobicity, have been identified, it is not well understood how the translocon and/or associated components decode them. Here we present evidence that the translocon-associated protein (TRAP) complex is involved in membrane protein topogenesis in vivo. Small interfering RNA (siRNA)-mediated silencing of the TRAP complex in HeLa cells enhanced the topology effect of mutating the flanking charges of a signal-anchor, but not of increasing signal hydrophobicity. The results suggest a role of the TRAP complex in moderating the ‘positive-inside’ rule.     

Transmembrane segments of nascent polytopic membrane proteins control cytosol/ER targeting during membrane integration

During cotranslational integration of a eukaryotic multispanning polytopic membrane protein (PMP), its hydrophilic loops are alternately directed to opposite sides of the ER membrane. Exposure of fluorescently labeled nascent PMP to the cytosol or ER lumen was detected by collisional quenching of its fluorescence by iodide ions localized in the cytosol or lumen. PMP loop exposure to the cytosol or lumen was controlled by structural rearrangements in the ribosome, translocon, and associated proteins that occurred soon after a nascent chain transmembrane segment (TMS) entered the ribosomal tunnel. Each successive TMS, although varying in length, sequence, hydrophobicity, and orientation, reversed the structural changes elicited by its predecessor, irrespective of loop size. Fluorescence lifetime data revealed that TMSs occupied a more nonpolar environment than secretory proteins inside the aqueous ribosome tunnel, which suggests that TMS recognition by the ribosome involves hydrophobic interactions. Importantly, the TMS-triggered structural rearrangements that cycle nascent chain exposure between cytosolic and lumenal occur without compromising the permeability barrier of the ER membrane.     

Sequential triage of transmembrane segments by Sec61alpha during biogenesis of a native multispanning membrane protein

During polytopic protein biogenesis, the Sec61 translocon must rapidly orient and integrate multiple transmembrane segments (TMs) into the endoplasmic reticulum membrane. To understand this process, we examined interactions between Sec61alpha and all six TMs of the aquaporin-4 (AQP4) water channel at defined stages of synthesis using incorporated photo-cross-linking probes. Each TM interacted with and moved through the translocon in a highly ordered and sequential fashion. Strong asymmetric Sec61alpha cross-linking was observed for only one helix at a time, suggesting the presence of a single primary binding site. However, up to four TMs simultaneously contacted Sec61alpha from different molecular environments. Thus, AQP4 integration by Sec61alpha involves sequential triage of TMs from their initial portal of entry into multiple secondary sites within the translocon. This mechanism provides a means to facilitate early folding events before release into the lipid bilayer.     

Systematic probing of the environment of a translocating secretory protein during translocation through the ER membrane.

We have extended a previously developed photo-crosslinking approach to systematically probe the protein environment of the secretory protein preprolactin, trapped during its transfer through the endoplasmic reticulum membrane. Single photoreactive groups were placed at various positions of nascent polypeptide chains of various length, corresponding to different stages of the transport process, and photo-crosslinks to membrane proteins were analyzed. In all cases, the polypeptide segment extending from the ribosome was found to be located in a membrane environment that is formed almost exclusively from Sec61 alpha, the multi-spanning subunit of the Sec61p complex that is essential for translocation. At early stages of the translocation process, before cleavage of the signal sequence, almost the entire nascent chain emerged from the ribosome contacts Sec61 alpha. The 'translocating chain-associating membrane' protein interacts mainly with the region of the signal sequence preceding its hydrophobic core. Our results suggest that the nascent chain is transferred directly from the ribosome into a protein-conducting channel, the major constituent of which is Sec61 alpha.     

Cooperation of transmembrane segments during the integration of a double-spanning protein into the ER membrane

While membrane insertion of single-spanning membrane proteins into the endoplasmic reticulum (ER) is relatively well understood, it is unclear how multi-spanning proteins integrate. We have investigated the cotranslational ER integration of a double-spanning protein that is derived from leader peptidase. Both transmembrane (TM) segments are inserted into the membrane by the Sec61 channel. While the first, long and hydrophobic TM segment (TM1) inserts into the lipid bilayer on its own, the second, shorter TM anchor (TM2) collaborates with TM1 during its integration. TM1 diffuses away from the Sec61 complex in the absence of TM2, but is close to Sec61 when TM2 arrives inside the channel. These data suggest that the exit of a weak TM segment from the Sec61 channel into the lipid phase can be facilitated by its interaction with a previously integrated strong and stabilizing TM anchor.     

Uncovering the membrane-integrated SecAN protein that plays a key role in translocating nascent outer membrane proteins

A large number of nascent polypeptides have to get across a membrane in targeting to the proper subcellular locations. The SecYEG protein complex, a homolog of the Sec61 complex in eukaryotic cells, has been viewed as the common translocon at the inner membrane for targeting proteins to three extracytoplasmic locations in Gram-negative bacteria, despite the lack of direct verification in living cells. Here, via unnatural amino acid-mediated protein-protein interaction analyses in living cells, in combination with genetic studies, we unveiled a hitherto unreported SecA^N protein that seems to be directly involved in translocationg nascent outer membrane proteins across the plasma membrane; it consists of the N-terminal 375 residues of the SecA protein and exists as a membrane-integrated homooligomer. Our new findings place multiple previous observations related to bacterial protein targeting in proper biochemical and evolutionary contexts.     

Different conformations of nascent polypeptides during translocation across the ER membrane

Background  

In eukaryotic cells, proteins are translocated across the ER membrane through a continuous ribosome-translocon channel. It is unclear to what extent proteins can fold already within the ribosome-translocon channel, and previous studies suggest that only a limited degree of folding (such as the formation of isolated α-helices) may be possible within the ribosome.     

Sec61alpha and TRAM are sequentially adjacent to a nascent viral membrane protein during its ER integration

Co-translational integration of a nascent viral membrane protein into the endoplasmic reticulum membrane takes place via the translocon. We have been studying the early stages of the integration of a double-spanning plant viral movement protein to gain insights into how viral membrane proteins are transferred from the hydrophilic interior of the translocon into the hydrophobic environment of the bilayer, where the transmembrane (TM) segments of the viral proteins can diffuse freely. Photocrosslinking experiments reveal that this integration involves the sequential passage of the TM segments past Sec61alpha and translocating chain-associating membrane protein (TRAM). Each TM segment is first adjacent to Sec61alpha and subsequently is adjacent to TRAM. TRAM crosslinking extends for a long period during nascent chain biogenesis. In addition, the replacement of the first viral TM segment with a non-viral TM sequence still yields nascent chain photo-adducts with TRAM. TRAM therefore appears to be involved in viral membrane protein integration, and nascent chain recognition by TRAM does not appear to rely solely on the TM domains.     

Reduced translocation of nascent prion protein during ER stress contributes to neurodegeneration

During acute stress in the endoplasmic reticulum (ER), mammalian prion protein (PrP) is temporarily prevented from translocation into the ER and instead routed directly for cytosolic degradation. This "pre-emptive" quality control (pQC) system benefits cells by minimizing PrP aggregation in the secretory pathway during ER stress. However, the potential toxicity of cytosolic PrP raised the possibility that persistent pQC of PrP contributes to neurodegeneration in prion diseases. Here, we find evidence of ER stress and decreased translocation of nascent PrP during prion infection. Transgenic mice expressing a PrP variant with reduced translocation at levels expected during ER stress was sufficient to cause several mild age-dependent clinical and histological manifestations of PrP-mediated neurodegeneration. Thus, an ordinarily adaptive quality-control pathway can be contextually detrimental over long time periods. We propose that one mechanism of prion-mediated neurodegeneration involves an indirect ER stress-dependent effect on nascent PrP biosynthesis and metabolism.     

A nascent membrane protein is located adjacent to ER membrane proteins throughout its integration and translation

The immediate environment of nascent membrane proteins undergoing integration into the ER membrane was investigated by photocrosslinking. Nascent polypeptides of different lengths, each containing a single IgM transmembrane sequence that functions either as a stop-transfer or a signal-anchor sequence, were synthesized by in vitro translation of truncated mRNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes. This yielded nascent chains with photoreactive probes at one end of the transmembrane sequence where two lysine residues are located. When irradiated, these nascent chains reacted covalently with several ER proteins. One prominent crosslinking target was a glycoprotein similar in size to a protein termed mp39, shown previously to be situated adjacent to a secretory protein during its translocation across the ER membrane (Krieg, U. C., A. E. Johnson, and P. Walter. 1989. J. Cell Biol. 109:2033-2043; Wiedmann, M., D. Goerlich, E. Hartmann, T. V. Kurzchalia, and T. A. Rapoport. 1989. FEBS (Fed. Eur. Biochem. Soc.) Lett. 257:263-268) and likely to be identical to a protein previously designated the signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature (Lond.). 328:830-833). Changing the orientation of the transmembrane domain in the bilayer, or making the transmembrane domain the first topogenic sequence in the nascent chain instead of the second, did not significantly alter the identities of the ER proteins that were the primary crosslinking targets. Furthermore, the nascent chains crosslinked to the mp39-like glycoprotein and other microsomal proteins even after the cytoplasmic tail of the nascent chain had been lengthened by nearly 100 amino acids beyond the stop-transfer sequence. Yet when the nascent chain was allowed to terminate normally, the major photocrosslinks were no longer observed, including in particular that to the mp39-like glycoprotein. These results show that the transmembrane segment of a nascent membrane protein is located adjacent to the mp39-like glycoprotein and other ER proteins during the integration process, and that at least a portion of the nascent chain remains in close proximity to these ER proteins until translation has been completed.     

Proper insertion and topogenesis of membrane proteins in the ER depend on Sec63

BackgroundIn eukaryotic cells, biogenesis of proteins destined to the secretory pathway begins from the cytosol. Nascent chains are either co-translationally or post-translationally targeted to the endoplasmic reticulum (ER) and translocated across the membrane through the Sec61 complex. For the post-translational translocation, the Sec62/Sec63 complex is additionally required. Sec63, however, is also shown to mediate co-translational translocation of a subset of proteins, the types and characteristics of proteins that Sec63 mediates in translocation still await to be defined.MethodsTo overview the types of proteins that require Sec63 for the ER translocation, we prepared Sec63 mutant lacking the first 39 residues (Sec63_ΔN39) in yeast and assessed initial translocation efficiencies of diverse types of precursors in the sec63_ΔN39 strain by a 5 min metabolic labeling. By employing Blue-Native gel electrophoresis (BN-PAGE), stability of the SEC complex (Sec61 plus Sec62/Sec63 complexes) isolated from cells carrying the Sec63_ΔN39 mutant was examined.ResultsAmong the various translocation precursors tested, we found that proper sorting of single- and double-pass membrane proteins was severely impaired in addition to post-translational translocation precursor in the sec63_ΔN39 mutant strain. Stability of the SEC complex was compromised upon deletion of the N-terminal 39 residues.ConclusionsThe N-terminus of Sec63 is important for stability of the SEC complex and Sec63 is required for proper sorting of membrane proteins in vivo.General significanceSec63 is essential on insertion of membrane proteins.     

Inter-helical hydrogen bond formation during membrane protein integration into the ER membrane

Recent work has shown that efficient di- or trimerization of hydrophobic transmembrane helices in detergent micelles or lipid bilayers can be driven by inter-helix hydrogen bonding involving polar residues such as Asn or Asp. Using in vitro translation in the presence of rough microsomes of a model integral membrane protein, we now show that the formation of so-called helical hairpins, two tightly spaced transmembrane helices connected by a short loop, can likewise be promoted by the introduction of Asn-Asn or Asp-Asp pairs in a long transmembrane hydrophobic segment. These observations suggest that inter-helix hydrogen bonds can form within the context of the Sec61 translocon in the endoplasmic reticulum, implying that hydrophobic segments in a nascent polypeptide chain in transit through the Sec61 channel have immediate access to a non-aqueous subcompartment within the translocon.     

Transmembrane topogenesis of a tail-anchored protein is modulated by membrane lipid composition

A large class of proteins with cytosolic functional domains is anchored to selected intracellular membranes by a single hydrophobic segment close to the C-terminus. Although such tail-anchored (TA) proteins are numerous, diverse, and functionally important, the mechanism of their transmembrane insertion and the basis of their membrane selectivity remain unclear. To address this problem, we have developed a highly specific, sensitive, and quantitative in vitro assay for the proper membrane-spanning topology of a model TA protein, cytochrome b5 (b5). Selective depletion from membranes of components involved in cotranslational protein translocation had no effect on either the efficiency or topology of b5 insertion. Indeed, the kinetics of transmembrane insertion into protein-free phospholipid vesicles was the same as for native ER microsomes. Remarkably, loading of either liposomes or microsomes with cholesterol to levels found in other membranes of the secretory pathway sharply and reversibly inhibited b5 transmembrane insertion. These results identify the minimal requirements for transmembrane topogenesis of a TA protein and suggest that selectivity among various intracellular compartments can be imparted by differences in their lipid composition.     

Cotranslational protein integration into the ER membrane is mediated by the binding of nascent chains to translocon proteins

During cotranslational protein integration into the ER membrane, each transmembrane (TM) segment moves laterally through the translocon to reach the lipid bilayer. Photocrosslinking studies reveal that a particular surface of each nascent chain TM alpha helix and signal-anchor sequence always faces Sec61alpha in the translocon. This nonrandom and TM sequence-dependent positioning reveals that each TM segment makes specific contacts with Sec61alpha and is retained at a fixed location within the translocon, observations that are best explained by the binding of each TM sequence to a translocon protein(s). Since TM sequence hydrophobicity does not correlate with its rate of release from the translocon, nascent chain movement through the translocon appears to be mediated primarily by protein-protein interactions rather than hydrophobic nascent chain-phospholipid interactions.     

Folding of the nascent peptide chain into a biologically active protein

The refolding of denatured proteins with complete sequences may not be fast enough to account for the in vivo folding of growing peptide chains during biosynthesis. As some peptide fragments have secondary structures not unlike those of the corresponding segments in the intact molecules and native disulfide bonds of some proteins can form cotranslationally, it is suggested that the folding of the nascent chain begins early during synthesis. However, further adjustments may be necessary during chain elongation and after posttranslational modifications of the completed peptide chain to generate the native conformation of a biologically active protein.     

Insertion of a multispanning membrane protein occurs sequentially and requires only one signal sequence

To study the insertion of multispanning membrane proteins into the endoplasmic reticulum, we constructed novel proteins on the cDNA level by repeating, up to four times, the internal signal-anchor domain of the asialoglycoprotein receptor H1. Upon in vitro translation in the presence of microsomes, these polypeptides are indeed inserted as polytopic membrane proteins. The first hydrophobic domain functions as a signal and the second as a stop-transfer sequence, while the third initiates a second translocation process, halted again by the fourth. We were able to demonstrate that insertion occurs sequentially, starting with the first apolar segment from the amino terminus. By replacing the original signal-anchor domains by a mutant sequence not recognized by signal recognition particle (SRP), it was shown that only the first hydrophobic domain needs to be a signal sequence and that the second translocation event does not require SRP.     

Glycosylation can influence topogenesis of membrane proteins and reveals dynamic reorientation of nascent polypeptides within the translocon.

The topology of multispanning membrane proteins in the mammalian endoplasmic reticulum is thought to be dictated primarily by the first hydrophobic sequence. We analyzed the in vivo insertion of a series of chimeric model proteins containing two conflicting signal sequences, i.e., an NH(2)-terminal and an internal signal, each of which normally directs translocation of its COOH-terminal end. When the signals were separated by more than 60 residues, linear insertion with the second signal acting as a stop-transfer sequence was observed. With shorter spacers, an increasing fraction of proteins inserted with a translocated COOH terminus as dictated by the second signal. Whether this resulted from membrane targeting via the second signal was tested by measuring the targeting efficiency of NH(2)-terminal signals followed by polypeptides of different lengths. The results show that targeting is mediated predominantly by the first signal in a protein. Most importantly, we discovered that glycosylation within the spacer sequence affects protein orientation. This indicates that the nascent polypeptide can reorient within the translocation machinery, a process that is blocked by glycosylation. Thus, topogenesis of membrane proteins is a dynamic process in which topogenic information of closely spaced signal and transmembrane sequences is integrated.     

A ribosome–nascent chain sensor of membrane protein biogenesis in Bacillus subtilis

Proteins in the YidC/Oxa1/Alb3 family have essential functions in membrane protein insertion and folding. Bacillus subtilis encodes two YidC homologs, one that is constitutively expressed (spoIIIJ/yidC1) and a second (yqjG/yidC2) that is induced in spoIIIJ mutants. Regulated induction of yidC2 allows B. subtilis to maintain capacity of the membrane protein insertion pathway. We here show that a gene located upstream of yidC2 (mifM/yqzJ) serves as a sensor of SpoIIIJ activity that regulates yidC2 translation. Decreased SpoIIIJ levels or deletion of the MifM transmembrane domain arrests mifM translation and unfolds an mRNA hairpin that otherwise blocks initiation of yidC2 translation. This regulated translational arrest and yidC2 induction require a specific interaction between the MifM C‐terminus and the ribosomal polypeptide exit tunnel. MifM therefore acts as a ribosome–nascent chain complex rather than as a fully synthesized protein. B. subtilis MifM and the previously described secretion monitor SecM in Escherichia coli thereby provide examples of the parallel evolution of two regulatory nascent chains that monitor different protein export pathways by a shared molecular mechanism.     

Substrate-specific function of the translocon-associated protein complex during translocation across the ER membrane

Although the transport of model proteins across the mammalian ER can be reconstituted with purified Sec61p complex, TRAM, and signal recognition particle receptor, some substrates, such as the prion protein (PrP), are inefficiently or improperly translocated using only these components. Here, we purify a factor needed for proper translocation of PrP and identify it as the translocon-associated protein (TRAP) complex. Surprisingly, TRAP also stimulates vectorial transport of many, but not all, other substrates in a manner influenced by their signal sequences. Comparative analyses of several natural signal sequences suggest that a dependence on TRAP for translocation is not due to any single physical parameter, such as hydrophobicity of the signal sequence. Instead, a functional property of the signal, efficiency of its post-targeting role in initiating substrate translocation, correlates inversely with TRAP dependence. Thus, maximal translocation independent of TRAP can only be achieved with a signal sequence, such as the one from prolactin, whose strong interaction with the translocon mediates translocon gating shortly after targeting. These results identify the TRAP complex as a functional component of the translocon and demonstrate that it acts in a substrate-specific manner to facilitate the initiation of protein translocation.     

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.