A stop transfer sequence recognizes receptors for nascent chain translocation across the endoplasmic reticulum membrane

A stop transfer sequence derived from the extreme carboxyl terminus of membrane IgM heavy chain has been shown to confer predictable transmembrane orientation to secretory proteins by aborting translocation of subsequently synthesized protein domains. Here we demonstrate that, in certain peptide sequence contexts, the same stop transfer sequence is also capable of initiating domain translocation across the endoplasmic reticulum (ER) membrane. Translocation directed by a stop transfer sequence is similar to, but distinguishable from, the action of a conventional signal sequence. Translocation is dependent on participation of the ribosome and protein receptors both in the cytoplasm and in the ER membrane. Moreover, both amino- and carboxy-terminal flanking protein domains can be translocated. Unlike a signal sequence, the stop transfer sequence is not itself translocated across the membrane. These results have implications for the nature of signal sequences, stop transfer sequences, and their receptor interactions.     

Pause transfer: a topogenic sequence in apolipoprotein B mediates stopping and restarting of translocation.

Previously, we described the stepwise translocation of a large amino-terminal fragment of apolipoprotein B (apo B15) in which the nascent secretory chain translocates through a series of distinct, nonintegrated transmembrane intermediates with large domains exposed to the cytoplasm. Thus, apo B15 appears to stop and restart translocation at several points. We have identified a sequence of amino acids in apo B15 that confers this behavior on a heterologous chimeric protein. In addition, we dissect pausing into two distinct steps, stopping and restarting, thereby trapping otherwise transient intermediates. Finally, we demonstrate the function of a second "pause transfer" sequence over 200 amino acids downstream in apo B15 that restarts translocation posttranslationally, suggesting that multiple pause transfer sequences are involved in the biogenesis of apolipoprotein B.     

Cooperativity and flexibility of cystic fibrosis transmembrane conductance regulator transmembrane segments participate in membrane localization of a charged residue

Polytopic protein topology is established in the endoplasmic reticulum (ER) by sequence determinants encoded throughout the nascent polypeptide. Here we characterize 12 topogenic determinants in the cystic fibrosis transmembrane conductance regulator, and identify a novel mechanism by which a charged residue is positioned within the plane of the lipid bilayer. During cystic fibrosis transmembrane conductance regulator biogenesis, topology of the C-terminal transmembrane domain (TMs 7-12) is directed by alternating signal (TMs 7, 9, and 11) and stop transfer (TMs 8, 10, and 12) sequences. Unlike conventional stop transfer sequences, however, TM8 is unable to independently terminate translocation due to the presence of a single charged residue, Asp(924), within the TM segment. Instead, TM8 stop transfer activity is specifically dependent on TM7, which functions both to initiate translocation and to compensate for the charged residue within TM8. Moreover, even in the presence of TM7, the N terminus of TM8 extends significantly into the ER lumen, suggesting a high degree of flexibility in establishing TM8 transmembrane boundaries. These studies demonstrate that signal sequences can markedly influence stop transfer behavior and indicate that ER translocation machinery simultaneously integrates information from multiple topogenic determinants as they are presented in rapid succession during polytopic protein biogenesis.     

Regulation of protein biogenesis at the endoplasmic reticulum membrane

The biogenesis of most secretory and membrane proteins involves targeting the nascent protein to the endoplasmic reticulum (ER), translocation across or integration into the ER membrane and maturation into a functional product. The essential machinery that directs these events for model secretory and membrane proteins has been identified, shifting the focus of studies towards the molecular mechanisms by which these core components function. By contrast, regulatory mechanisms that allow certain proteins to serve multiple functions within a cell remain entirely unexplored. This article examines each stage of protein biogenesis as a potential site of regulation that could be exploited by the cell to effectively increase the diversity of functional gene expression.     

Protection from cytosolic prion protein toxicity by modulation of protein translocation

Failure to promptly dispose of undesirable proteins is associated with numerous diseases. In the case of cellular prion protein (PrP), inhibition of the proteasome pathway can generate a highly aggregation-prone, cytotoxic form of PrP implicated in neurodegeneration. However, the predominant mechanisms that result in delivery of PrP, ordinarily targeted to the secretory pathway, to cytosolic proteasomes have been unclear. By accurately measuring the in vivo fidelity of protein translocation into the endoplasmic reticulum (ER), we reveal a slight inefficiency in PrP signal sequence function that generates proteasomally degraded cytosolic PrP. Attenuating this source of cytosolic PrP completely eliminates the dependence on proteasomes for PrP degradation. This allows cells to tolerate both higher expression levels and decreased proteasomal capacity without succumbing to the adverse consequences of misfolded PrP. Thus, the generation of potentially toxic cytosolic PrP is controlled primarily during its initial translocation into the ER. These results suggest that a substantial proportion of the cell's constitutive proteasomal burden may consist of proteins that, like PrP, fail to cotranslationally enter the secretory pathway with high fidelity.     

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.     

Substrate-specific translocational attenuation during ER stress defines a pre-emptive quality control pathway

Eukaryotic proteins entering the secretory pathway are translocated into the ER by signal sequences that vary widely in primary structure. We now provide a functional rationale for this long-observed sequence diversity by demonstrating that differences among signals facilitate substrate-selective modulation of protein translocation. We find that during acute ER stress, translocation of secretory and membrane proteins is rapidly and transiently attenuated in a signal sequence-selective manner. Their cotranslational rerouting to the cytosol for degradation reduces the burden of misfolded substrates entering the ER and represents a pathway for pre-emptive quality control (pQC). Bypassing the pQC pathway for the prion protein increases its rate of aggregation in the ER lumen during prolonged stress and renders cells less capable of viable recovery. Conversely, pharmacologically augmenting pQC during ER stress proved protective. Thus, protein translocation is a physiologically regulated process that is utilized for pQC as part of the ER stress response.     

Export of a cysteine-free misfolded secretory protein from the endoplasmic reticulum for degradation requires interaction with protein disulfide isomerase

Protein disulfide isomerase (PDI) interacts with secretory proteins, irrespective of their thiol content, late during translocation into the ER; thus, PDI may be part of the quality control machinery in the ER. We used yeast pdi1 mutants with deletions in the putative peptide binding region of the molecule to investigate its role in the recognition of misfolded secretory proteins in the ER and their export to the cytosol for degradation. Our pdi1 deletion mutants are deficient in the export of a misfolded cysteine-free secretory protein across the ER membrane to the cytosol for degradation, but ER-to-Golgi complex transport of properly folded secretory proteins is only marginally affected. We demonstrate by chemical cross-linking that PDI specifically interacts with the misfolded secretory protein and that mutant forms of PDI have a lower affinity for this protein. In the ER of the pdi1 mutants, a higher proportion of the misfolded secretory protein remains associated with BiP, and in export-deficient sec61 mutants, the misfolded secretory protein remain bounds to PDI. We conclude that the chaperone PDI is part of the quality control machinery in the ER that recognizes terminally misfolded secretory proteins and targets them to the export channel in the ER membrane.     

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.     

Evidence for a two-step mechanism involved in assembly of functional signal recognition particle receptor

The signal recognition particle (SRP) and SRP receptor act sequentially to target nascent secretory proteins to the membrane of the ER. The SRP receptor consists of two subunits, SR alpha and SR beta, both tightly associated with the ER membrane. To examine the biogenesis of the SRP receptor we have developed a cell-free assay system that reconstitutes SR alpha membrane assembly and permits both anchoring and functional properties to be assayed independently. Our experiments reveal a mechanism involving at least two distinct steps, targeting to the ER and anchoring of the targeted molecule on the cytoplasmic face of the membrane. Both steps can be reconstituted in vitro to restore translocation activity to ER microsomes inactivated by alkylation with N-ethyl-maleimide. The characteristics elucidated for this pathway distinguish it from SRP-dependent targeting of secretory proteins, SRP-independent ER translocation of proteins such as prepromellitin, and direct insertion mechanisms of the type exemplified by cytochrome b5.     

Getting greasy: how transmembrane polypeptide segments integrate into the lipid bilayer

Many integral membrane proteins use the same translocation machinery for membrane insertion as secretory proteins use to get across the membrane. This requires that transmembrane segments can be discriminated from other parts of the protein during membrane translocation, and further requires that the transmembrane segments can be moved laterally out of the translocation channel into the surrounding lipid. The molecular basis for this remarkable intramembraneous sorting event is a major focus of current studies of membrane protein biogenesis.     

Identification of sequence determinants that direct different intracellular folding pathways for aquaporin-1 and aquaporin-4

Homologous aquaporin water channels utilize different folding pathways to acquire their transmembrane (TM) topology in the endoplasmic reticulum (ER). AQP4 acquires each of its six TM segments via cotranslational translocation events, whereas AQP1 is initially synthesized with four TM segments and subsequently converted into a six membrane-spanning topology. To identify sequence determinants responsible for these pathways, peptide segments from AQP1 and AQP4 were systematically exchanged. Chimeric proteins were then truncated, fused to a C-terminal translocation reporter, and topology was analyzed by protease accessibility. In each chimeric context, TM1 initiated ER targeting and translocation. However, AQP4-TM2 cotranslationally terminated translocation, while AQP1-TM2 failed to terminate translocation and passed into the ER lumen. This difference in stop transfer activity was due to two residues that altered both the length and hydrophobicity of TM2 (Asn(49) and Lys(51) in AQP1 versus Met(48) and Leu(50) in AQP4). A second peptide region was identified within the TM3-4 peptide loop that enabled AQP4-TM3 but not AQP1-TM3 to reinitiate translocation and cotranslationally span the membrane. Based on these findings, it was possible to convert AQP1 into a cotranslational biogenesis mode similar to that of AQP4 by substituting just two peptide regions at the N terminus of TM2 and the C terminus of TM3. Interestingly, each of these substitutions disrupted water channel activity. These data thus establish the structural basis for different AQP folding pathways and provide evidence that variations in cotranslational folding enable polytopic proteins to acquire and/or maintain primary sequence determinants necessary for function.     

Prion protein contains a second endoplasmic reticulum targeting signal sequence located at its C terminus

Prion protein (PrP) is synthesized at the membrane of the endoplasmic reticulum (ER) in three different topological forms as follows: a fully translocated one ((sec)PrP) and two with opposite orientations in the membrane ((Ntm)PrP and (Ctm)PrP). We asked whether other signal sequences exist in the PrP, other than the N-terminal signal sequence, that contribute to its topological diversity. In vitro translocation assays showed that PrP lacking its N-terminal signal sequence could still translocate into ER microsomes, although at reduced efficiency. Deletion of each of the two hydrophobic regions in PrP revealed that the C-terminally located hydrophobic region (TM2) can function as second signal sequence in PrP. Translocation mediated by the TM2 alone can occur post-translationally and yields mainly (Ctm)PrP, which is implicated in some forms of neurodegeneration in prion diseases. We conclude that, in vitro, PrP can insert into ER membranes co- and post-translationally and can use two different signal sequences. We propose that the unusually complex topology of PrP results from the differential utilization of two signal sequences in PrP.     

A calmodulin-dependent translocation pathway for small secretory proteins

Metazoans secrete an extensive array of small proteins essential for intercellular communication, defense, and physiologic regulation. Their synthesis takes mere seconds, leaving minimal time for recognition by the machinery for cotranslational protein translocation into the ER. The pathway taken by these substrates to enter the ER is not known. Here, we show that both in vivo and in vitro, small secretory proteins can enter the ER posttranslationally via a transient cytosolic intermediate. This intermediate contained calmodulin selectively bound to the signal peptides of small secretory proteins. Calmodulin maintained the translocation competence of small-protein precursors, precluded their aggregation and degradation, and minimized their inappropriate interactions with other cytosolic polypeptide-binding proteins. Acute inhibition of calmodulin specifically impaired small-protein translocation in vitro and in cells. These findings establish a mammalian posttranslational pathway for small-protein secretion and identify an unexpected role for calmodulin in chaperoning these precursors safely through the cytosol.     

Stop-transfer activity of hydrophobic sequences depends on the translation system

Signal and stop-transfer sequences are the known determinants involved in topogenesis of integral membrane proteins. To study the characteristics of stop-transfer sequences, artificial proteins have been created on the DNA level based on the cDNA of the asialoglycoprotein receptor H1. Its internal signal/anchor domain initiates translocation of the downstream sequence across the endoplasmic reticulum membrane. The ability of several hydrophobic sequences inserted into the translocating polypeptide to stop further transfer was analyzed by translation of the fusion proteins using the wheat germ extract and rabbit reticulocyte lysate systems with dog pancreas microsomes. We discovered that some of the sequences behave differently with respect to translocation across the membrane depending on the translation system. Expression of one of the fusion proteins in fibroblasts showed that the reticulocyte lysate system reflects more closely the in vivo situation than the wheat germ system. Our results suggest that in a homologous system the translating ribosomes interact with the translocation machinery and influence the termination of polypeptide transfer by hydrophobic sequences.     

The stop transfer sequence of the human UDP-glucuronosyltransferase 1A determines localization to the endoplasmic reticulum by both static retention and retrieval mechanisms

Human UDP-glucuronosyltransferase 1A (UGT1A) isoforms are endoplasmic reticulum (ER)-resident type I membrane proteins responsible for the detoxification of a broad range of toxic phenolic compounds. These proteins contain a C-terminal stop transfer sequence with a transmembrane domain (TMD), which anchors the protein into the membrane, followed by a short cytosolic tail (CT). Here, we investigated the mechanism of ER residency of UGT1A mediated by the stop transfer sequence by analysing the subcellular localization and sensitivity to endoglycosidases of chimeric proteins formed by fusion of UGT1A stop transfer sequence (TMD/CT) with the ectodomain of the plasma membrane CD4 reporter protein. We showed that the stop transfer sequence, when attached to C-terminus of the CD4 ectodomain was able to prevent it from being transported to the cell surface. The protein was retained in the ER indicating that this sequence functions as an ER localization signal. Furthermore, we demonstrated that ER localization conferred by the stop transfer sequence was mediated in part by the KSKTH retrieval signal located on the CT. Interestingly, our data indicated that UGT1A TMD alone was sufficient to retain the protein in ER without recycling from Golgi compartment, and brought evidence that organelle localization conferred by UGT1A TMD was determined by the length of its hydrophobic core. We conclude that both retrieval mechanism and static retention mediated by the stop transfer sequence contribute to ER residency of UGT1A proteins.     

Structural requirements for membrane assembly of proteins spanning the membrane several times

We have investigated the structural requirements for the biogenesis of proteins spanning the membrane several times. Proteins containing various combinations of topological signals (signal anchor and stop transfer sequences) were synthesized in a cell-free translation system and their membrane topology was determined. Proteins spanning the membrane twice were obtained when a signal anchor sequence was followed by either a stop transfer sequence or a second signal anchor sequence. Thus, a signal anchor sequence in the second position can function as a stop transfer sequence, spanning the membrane in the opposite orientation to that of the first signal anchor sequence. A signal anchor sequence in the third position was able to insert amino acid sequences located COOH terminal to it. We conclude that proteins spanning the membrane several times can be generated by stringing together signal anchor and stop transfer sequences. However, not all proteins with three topological signals were found to span the membrane three times. A certain segment located between the first and second topological signal could prevent stable membrane integration of a third signal anchor segment.     

Sec61p and BiP directly facilitate polypeptide translocation into the ER.

Secretory proteins are segregated from cytosolic proteins by their translocation into the endoplasmic reticulum (ER). A modified secretory protein trapped during translocation across the ER membrane can be crosslinked to two previously identified proteins, Sec61p and BiP (Kar2p). The dependence of this cross-linking upon proteins and small molecules was examined. Mutations in SEC62 and SEC63 decrease the ability of Sec61p to be cross-linked to the secretory polypeptide trapped in translocation. ATP is also required for interaction of Sec61p with the secretory protein. Three kar2 alleles display defective translocation in vitro. Two of these alleles also decrease the ability of Sec61p to be cross-linked to the secretory protein. The third allele, while exhibiting a severe translocation defect, does not affect the interaction of Sec61p with the secretory protein. These results suggest that Sec61p is directly involved in translocation and that BiP acts at two stages of the translocation cycle.     

TorsinA folding and N-linked glycosylation are sensitive to redox homeostasis

The Endoplasmic Reticulum (ER) is responsible for the folding and post-translational modification of secretory proteins, as well as for triaging misfolded proteins. During folding, there is a complex yet only partially understood interplay between disulfide bond formation, which is an enzyme catalyzed event in the oxidizing environment of the ER, along with other post-translational modifications (PTMs) and chaperone-supported protein folding. Here, we used the glycoprotein torsinA as a model substrate to explore the impact of ER redox homeostasis on PTMs and protein biogenesis. TorsinA is a AAA+ ATPase with unusual oligomeric properties and controversial functions. The deletion of a C-terminal glutamic acid residue (?E) is associated with the development of Early-Onset Torsion Dystonia, a severe movement disorder. TorsinA differs from other AAA+ ATPases since it is an ER resident, and as a result of its entry into the ER torsinA contains two N-linked glycans and at least one disulfide bond. The role of these PTMs on torsinA biogenesis and function and the identity of the enzymes that catalyze them are poorly defined. Using a yeast torsinA expression system, we demonstrate that a specific protein disulfide isomerase, Pdi1, affects the folding and N-linked glycosylation of torsinA and torsinA?E in a redox-dependent manner, suggesting that the acquisition of early torsinA folding intermediates is sensitive to perturbed interactions between Cys residues and the quality control machinery. We also highlight the role of specific Cys residues during torsinA biogenesis and demonstrate that torsinA?E is more sensitive than torsinA when these Cys residues are mutated.