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.     

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.     

Protein-specific features of the general secretion pathway in yeast: the secretion of acid phosphatase

The major phosphate-repressible acid phosphatase (APase) of Saccharomyces cerevisiae, a cell wall glycoprotein, has been extensively used as a reporter protein to analyse successive steps in the yeast secretory pathway. In contrast to other yeast secretory proteins, APase can still be translocated into the endoplasmic reticulum (ER) even when it is made without its signal peptide. This property illustrates the permissiveness of targeting to the ER in yeast. Studies on APase-containing hybrid proteins have provided some of the evidence that specific soluble factors must interact with secretory proteins prior to their translocation across the ER membrane. A systematic analysis of mutations affecting the sequence of the APase signal peptide cleavage site demonstrated that cleavage occurs only when the last amino acid of the signal sequence is small and neutral. This was one of the first studies to verify the requirements for signal peptidase cleavage that had previously only been predicted from statistical analysis. Studies performed either with inhibitors of glycosylation or with mutant APases demonstrated the critical role of core glycosylation for APase folding, which is essential for efficient transport beyond the ER. Following the fate of particular modified APases along the secretory pathway provided insights into some general properties of the secretory apparatus and illustrated the specific requirements for a given protein during its intracellular traffic.     

Bimodal targeting of cytochrome P450s to endoplasmic reticulum and mitochondria: the concept of chimeric signals

Targeting signals are critical for proteins to find their specific cellular destination. Signals for protein targeting to the endoplasmic reticulum (ER), mitochondria, peroxisome and nucleus are distinct and the mechanisms of protein translocation across these membrane compartments also vary markedly. Recently, however, a number of proteins have been shown to be present in multiple cellular sites such as mitochondria and ER, cytosol and mitochondria, plasma membrane and mitochondria, and peroxisome and mitochondria suggesting the occurrence of multimodal targeting signals in some cases. Cytochrome P450 monooxygenases (CYPs), which play crucial roles in pharmacokinetics and pharmacodynamics of drugs and toxins, are the prototype of bimodally targeted proteins. Several members of family 1, 2 and 3 CYPs have now been reported to be associated with mitochondria and plasma membrane in addition to the ER. This review highlights the mechanisms of bimodal targeting of CYP1A1, 2B1, 2E1 and 2D6 to mitochondria and ER. The bimodal targeting of these proteins is driven by their N-terminal signals which carry essential elements of both ER targeting and mitochondria targeting signals. These multimodal signals have been termed chimeric signals appropriately to describe their dual targeting property. The cryptic mitochondrial targeting signals of CYP2B1, 2D6, 2E1 require activation by protein kinase A or protein kinase C mediated phosphorylation at sites immediately flanking the targeting signal and/or membrane anchoring regions. The cryptic mitochondria targeting signal of CYP1A1 requires activation by endoproteolytic cleavage by a cytosolic endoprotease, which exposes the mitochondrial signal. This review discusses both mechanisms of bimodal targeting and toxicological consequences of mitochondria targeted CYP proteins.     

Evidence for Golgi-independent transport from the early secretory pathway to the plastid in malaria parasites

The malaria parasite Plasmodium falciparum harbours a relict plastid (termed the apicoplast) that has evolved by secondary endosymbiosis. The apicoplast is surrounded by four membranes, the outermost of which is believed to be part of the endomembrane system. Nuclear-encoded apicoplast proteins have a two-part N-terminal extension that is necessary and sufficient for translocation across these four membranes. The first domain of this N-terminal extension resembles a classical signal peptide and mediates translocation into the secretory pathway, whereas the second domain is homologous to plant chloroplast transit peptides and is required for the remaining steps of apicoplast targeting. We explored the initial, secretory pathway component of this targeting process using green fluorescent reporter protein constructs with modified leaders. We exchanged the apicoplast signal peptide with signal peptides from other secretory proteins and observed correct targeting, demonstrating that apicoplast targeting is initiated at the general secretory pathway of P. falciparum. Furthermore, we demonstrate by immunofluorescent labelling that the apicoplast resides on a small extension of the endoplasmic reticulum (ER) that is separate from the cis-Golgi. To define the position of the apicoplast in the endomembrane pathway in relation to the Golgi we tracked apicoplast protein targeting in the presence of the secretory inhibitor Brefeldin A (BFA), which blocks traffic between the ER and Golgi. We observe apicoplast targeting in the presence of BFA despite clear perturbation of ER to Golgi traffic by the inhibitor, which suggests that the apicoplast resides upstream of the cis-Golgi in the parasite's endomembrane system. The addition of an ER retrieval signal (SDEL) - a sequence recognized by the cis-Golgi protein ERD2 - to the C-terminus of an apicoplast-targeted protein did not markedly affect apicoplast targeting, further demonstrating that the apicoplast is upstream of the Golgi. Apicoplast transit peptides are thus dominant over an ER retention signal. However, when the transit peptide is rendered non-functional (by two point mutations or by complete deletion) SDEL-specific ER retrieval takes over, and the fusion protein is localized to the ER. We speculate either that the apicoplast in P. falciparum resides within the ER directly in the path of the general secretory pathway, or that vesicular trafficking to the apicoplast directly exits the ER.     

All roads lead to Rome (but some may be harder to travel): SRP-independent translocation into the endoplasmic reticulum

Abstract

Translocation into the endoplasmic reticulum (ER) is the first biogenesis step for hundreds of eukaryotic secretome proteins. Over the past 30 years, groundbreaking biochemical, structural and genetic studies have delineated one conserved pathway that enables ER translocation- the signal recognition particle (SRP) pathway. However, it is clear that this is not the only pathway which can mediate ER targeting and insertion. In fact, over the past decade, several SRP-independent pathways have been uncovered, which recognize proteins that cannot engage the SRP and ensure their subsequent translocation into the ER. These SRP-independent pathways face the same challenges that the SRP pathway overcomes: chaperoning the preinserted protein while in the cytosol, targeting it rapidly to the ER surface and generating vectorial movement that inserts the protein into the ER. This review strives to summarize the various mechanisms and machineries which mediate these stages of SRP-independent translocation, as well as examine why SRP-independent translocation is utilized by the cell. This emerging understanding of the various pathways utilized by secretory proteins to insert into the ER draws light to the complexity of the translocational task, and underlines that insertion into the ER might be more varied and tailored than previously appreciated.     

Mutants in three novel complementation groups inhibit membrane protein insertion into and soluble protein translocation across the endoplasmic reticulum membrane of Saccharomyces cerevisiae

We have isolated mutants that inhibit membrane protein insertion into the ER membrane of Saccharomyces cerevisiae. The mutants were contained in three complementation groups, which we have named SEC70, SEC71, and SEC72. The mutants also inhibited the translocation of soluble proteins into the lumen of the ER, indicating that they pleiotropically affect protein transport across and insertion into the ER membrane. Surprisingly, the mutants inhibited the translocation and insertion of different proteins to drastically different degrees. We have also shown that mutations in SEC61 and SEC63, which were previously isolated as mutants inhibiting the translocation of soluble proteins, also affect the insertion of membrane proteins into the ER. Taken together our data indicate that the process of protein translocation across the ER membrane involves a much larger number of gene products than previously appreciated. Moreover, different translocation substrates appear to have different requirements for components of the cellular targeting and translocation apparatus.     

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.     

Human autoantibodies against the 54 kDa protein of the signal recognition particle block function at multiple stages

The 54 kDa subunit of the signal recognition particle (SRP54) binds to the signal sequences of nascent secretory and membrane proteins and it contributes to the targeting of these precursors to the membrane of the endoplasmic reticulum (ER). At the ER membrane, the binding of the signal recognition particle (SRP) to its receptor triggers the release of SRP54 from its bound signal sequence and the nascent polypeptide is transferred to the Sec61 translocon for insertion into, or translocation across, the ER membrane. In the current article, we have characterized the specificity of anti-SRP54 autoantibodies, which are highly characteristic of polymyositis patients, and investigated the effect of these autoantibodies on the SRP function in vitro. We found that the anti-SRP54 autoantibodies had a pronounced and specific inhibitory effect upon the translocation of the secretory protein preprolactin when analysed using a cell-free system. Our mapping studies showed that the anti-SRP54 autoantibodies bind to the amino-terminal SRP54 N-domain and to the central SRP54 G-domain, but do not bind to the carboxy-terminal M-domain that is known to bind ER signal sequences. Nevertheless, anti-SRP54 autoantibodies interfere with signal-sequence binding to SRP54, most probably by steric hindrance. When the effect of anti-SRP autoantibodies on protein targeting the ER membrane was further investigated, we found that the autoantibodies prevent the SRP receptor-mediated release of ER signal sequences from the SRP54 subunit. This observation supports a model where the binding of the homologous GTPase domains of SRP54 and the α-subunit of the SRP receptor to each other regulates the release of ER signal sequences from the SRP54 M-domain.     

Signal Recognition Particle and SecA Cooperate during Export of Secretory Proteins with Highly Hydrophobic Signal Sequences

The Sec translocon of bacterial plasma membranes mediates the linear translocation of secretory proteins as well as the lateral integration of membrane proteins. Integration of many membrane proteins occurs co-translationally via the signal recognition particle (SRP)-dependent targeting of ribosome-associated nascent chains to the Sec translocon. In contrast, translocation of classical secretory proteins across the Sec translocon is a post-translational event requiring no SRP but the motor protein SecA. Secretory proteins were, however, reported to utilize SRP in addition to SecA, if the hydrophobicity of their signal sequences exceeds a certain threshold value. Here we have analyzed transport of this subgroup of secretory proteins across the Sec translocon employing an entirely defined in vitro system. We thus found SecA to be both necessary and sufficient for translocation of secretory proteins with hydrophobic signal sequences, whereas SRP and its receptor improved translocation efficiency. This SRP-mediated boost of translocation is likely due to the early capture of the hydrophobic signal sequence by SRP as revealed by site-specific photo cross-linking of ribosome nascent chain complexes.     

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.     

A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum

We have devised a genetic selection for mutant yeast cells that fail to translocate secretory protein precursors into the lumen of the endoplasmic reticulum (ER). Mutant cells are selected by a procedure that requires a signal peptide-containing cytoplasmic enzyme chimera to remain in contact with the cytosol. This approach has uncovered a new secretory mutant, sec61, that is thermosensitive for growth and that accumulates multiple secretory and vacuolar precursor proteins that have not acquired any detectable posttranslational modifications associated with translocation into the ER. Preproteins that accumulate at the sec61 block sediment with the particulate fraction, but are exposed to the cytosol as judged by sensitivity to proteinase K. Thus, the sec61 mutation defines a gene that is required for an early cytoplasmic or ER membrane-associated step in protein translocation.     

Inefficient Translocation of Preproinsulin Contributes to Pancreatic β Cell Failure and Late-onset Diabetes

Among the defects in the early events of insulin biosynthesis, proinsulin misfolding and endoplasmic reticulum (ER) stress have drawn increasing attention as causes of β cell failure. However, no studies have yet addressed potential defects at the cytosolic entry point of preproinsulin into the secretory pathway. Here, we provide the first evidence that inefficient translocation of preproinsulin (caused by loss of a positive charge in the n region of its signal sequence) contributes to β cell failure and diabetes. Specifically, we find that, after targeting to the ER membrane, preproinsulin signal peptide (SP) mutants associated with autosomal dominant late-onset diabetes fail to be fully translocated across the ER membrane. The newly synthesized, untranslocated preproinsulin remains strongly associated with the ER membrane, exposing its proinsulin moiety to the cytosol. Rather than accumulating in the ER and inducing ER stress, untranslocated preproinsulin accumulates in a juxtanuclear compartment distinct from the Golgi complex, induces the expression of heat shock protein 70 (HSP70), and promotes β cell death. Restoring an N-terminal positive charge to the mutant preproinsulin SP significantly improves the translocation defect. These findings not only reveal a novel molecular pathogenesis of β cell failure and diabetes but also provide the first evidence of the physiological and pathological significance of the SP n region positive charge of secretory proteins.     

Endoplasmic reticulum composition and form: Proteins in and out

The endoplasmic reticulum (ER) is the main harbor for newly synthesized proteins in eukaryotic cells. Through a continuous membrane network of sheets and tubules, the ER hosts secretory proteins, integral membrane proteins, and luminal proteins of the endomembrane system. These proteins are translated by ribosomes outside the ER and require subsequent integration into or translocation across the lipid bilayer of the ER. They are then modified post-translationally and folded in the ER. Some of these proteins are packaged into coat protein complex II–coated vesicles for export. Here, we review recent advances in understanding the mechanism of protein translocation and transmembrane domain insertion in the ER, summarize new insights into selective cargo packaging, and discuss the roles of ER morphological dynamics in these processes.     

Targeting and translocation of two lipoproteins in Escherichia coli via the SRP/Sec/YidC pathway

In Escherichia coli, two main protein targeting pathways to the inner membrane exist: the SecB pathway for the essentially posttranslational targeting of secretory proteins and the SRP pathway for cotranslational targeting of inner membrane proteins (IMPs). At the inner membrane both pathways converge at the Sec translocase, which is capable of both linear transport into the periplasm and lateral transport into the lipid bilayer. The Sec-associated YidC appears to assist the lateral transport of IMPs from the Sec translocase into the lipid bilayer. It should be noted that targeting and translocation of only a handful of secretory proteins and IMPs have been studied. These model proteins do not include lipoproteins. Here, we have studied the targeting and translocation of two secretory lipoproteins, the murein lipoprotein and the bacteriocin release protein, using a combined in vivo and in vitro approach. The data indicate that both murein lipoprotein and bacteriocin release protein require the SRP pathway for efficient targeting to the Sec translocase. Furthermore, we show that YidC plays an important role in the targeting/translocation of both lipoproteins.     

Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane--distinct translocases and mechanisms

In bacteria, two major pathways exist to secrete proteins across the cytoplasmic membrane. The general Secretion route, termed Sec-pathway, catalyzes the transmembrane translocation of proteins in their unfolded conformation, whereupon they fold into their native structure at the trans-side of the membrane. The Twin-arginine translocation pathway, termed Tat-pathway, catalyses the translocation of secretory proteins in their folded state. Although the targeting signals that direct secretory proteins to these pathways show a high degree of similarity, the translocation mechanisms and translocases involved are vastly different.     

Regulation of ribosome detachment from the mammalian endoplasmic reticulum membrane

In current models, protein translocation in the endoplasmic reticulum (ER) occurs in the context of two cycles, the signal recognition particle (SRP) cycle and the ribosome cycle. Both SRP and ribosomes bind to the ER membrane as a consequence of the targeting process of translocation. Whereas SRP release from the ER membrane is regulated by the GTPase activities of SRP and the SRP receptor, ribosome release from the ER membrane is thought to occur in response to the termination of protein synthesis. We report that ER-bound ribosomes remain membrane-bound following the termination of protein synthesis and in the bound state can initiate the translation of secretory and cytoplasmic proteins. Two principal observations are reported. 1) Membrane-bound ribosomes engaged in the synthesis of proteins lacking a signal sequence are released from the ER membrane as ribosome-nascent polypeptide complexes. 2) Membrane-bound ribosomes translating secretory proteins can access the translocon in an SRP receptor-independent manner. We propose that ribosome release from the ER membrane occurs in the context of protein translation, with release occurring by default in the absence of productive nascent polypeptide-membrane interactions.     

Transmembrane movement of dolichol linked carbohydrates during N-glycoprotein biosynthesis in the endoplasmic reticulum

The process of N-linked glycosylation of secretory proteins is characterized by enzymatic reactions occurring on both sides of the endoplasmic reticulum (ER) membrane. On either side multiple glycosyltransferases participate in the stepwise addition of monosaccharides to core oligosaccharide unit that is attached to the lipid carrier dolichyl pyrophosphate. Cytoplasm-oriented glycosyltransferases use nucleotide-activated sugars as substrates, whereas lumen-oriented transferases that act later in the pathway make use of dolichyl phosphate-linked monosaccharides. The completely assembled core oligosaccharide is transferred to proteins on the lumenal side of the ER. The topological organization of this biosynthetic pathway requires the translocation of lipid-linked mono- and oligo-saccharides across the ER membrane. The transfer of the substrates and intermediates depend on specific translocators, i.e. so called flippases.