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Protein Secretion and the Endoplasmic Reticulum Downloaded from http://cshperspectives.cshlp.org/ on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press Protein Secretion and the Endoplasmic Reticulum Adam M. Benham School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, United Kingdom Correspondence: [email protected] In a complex multicellular organism, different cell types engage in specialist functions, and as a result, the secretory output of cells and tissues varies widely. Whereas some quiescent cell types secrete minor amounts of proteins, tissues like the pancreas, producing insulin and other hormones, and mature B cells, producing antibodies, place a great demand on their endoplasmic reticulum (ER). Our understanding of how protein secretion in general is con- trolled in the ER is now quite sophisticated. However, there remain gaps in our knowledge, particularly when applying insight gained from model systems to the more complex situa- tions found in vivo. This article describes recent advances in our understanding of the ER and its role in preparing proteins for secretion, with an emphasis on glycoprotein quality control and pathways of disulfide bond formation. ow a cell controls its complex output and the folding and quality control of the protein, Hensures that only properly folded and func- the availability of cofactors (particularly calci- tional proteins reach their correct destination is um), and the appropriate formation of disulfide a question of considerable importance in biol- bonds (Braakman and Bulleid 2011). Some pro- ogy. It has been calculated that the products of teins require assembly into complexes, and pro- 11% of the approximately 25,000 predicted hu- vision of lipids for ER membranes and the reg- man full-length open reading frames (ORFs) are ulation of cholesterol content are also important soluble secretory proteins, with a further 20% considerations. Here, the focus is on recent ad- being single-pass or multi-pass transmembrane vances in our understanding of the glycosylation proteins that get targeted to the ER (Kanapin machinery, the provision of disulfide bonds, and et al. 2003). Many of these proteins can also be the ER exit strategies used by proteins to ensure alternatively spliced, adding a further layer of their efficient secretion from the ER (Fig. 1). complexity (Carninci et al. 2005). To ensure or- derly ER exit, protein secretion from the ER is TARGETING OF PROTEINS TO THE ER determined byseveral factors. These include tar- geting of the nascent protein to the ER by the In eukaryotic cells, most secreted proteins are ribosome, the translocation of the protein into cotranslationally translocated from ribosomes the ER, the provision of sugars for glycoproteins, into the rough ER. However, some proteins, Editors: John W.B. Hershey, Nahum Sonenberg, and Michael B. Mathews Additional Perspectives on Protein Synthesis and Translational Control available at www.cshperspectives.org Copyright # 2012 Cold Spring Harbor Laboratory Press; all rights reserved. Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a012872 1 Downloaded from http://cshperspectives.cshlp.org/ on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press A.M. Benham 3 2 Ribosome OST 1 Nascent protein N-linked glycan SRP Translocon SRP receptor Disulfide oxido- Disulfide reductase oxido- reductase S-S Calnexin Cytosol ER cycle 54 6 S-S ERGIC Figure 1. Overview of protein targeting to the ER and glycoprotein quality control. Six key points in the fidelity of protein secretion are illustrated. (1) Correct targeting of the glycoprotein to the ER as it emerges from the translocon. This is mediated by the signal recognition particle (SRP) and its receptor, which helps position the emerging protein at the translocon. (2) Translocation of the (glyco)protein into the ER by the translocon. (3) Addition of N-glycans to Asn residues of glycoproteins by oligosaccharyl transferase (OST). (4) Correct folding and quality control by the calnexin cycle. (5) Introduction/correction of disulfide bonds by protein disulfide isomerases/oxidoreductases. (6) Directing glycoproteins into ER exit sites followed by packaging into appropriate compartments, for example, the ER Golgi intermediate compartment (ERGIC). Note that protein folding and disulfide bond formation happen rapidly after the nascent protein emerges from the translocon. (S–S) Disulfide bond; (A) N-Acetyl glucosamine; (W) mannose; (4) glucose. particularly small polypeptides or proteins with particle (SRP) (Saraogi and Shan 2011). SRP is “weak” signal sequences, can access the ER post- composed of six proteins (P9/P14, P68/P72, translationally (Ng et al. 1996; Shao and Hegde P19, and P54) and a 300-nucleotide RNA scaf- 2011). The cotranslational targeting of secreted fold. The P54 subunit of SRP is primarily re- proteins to the ER is achieved by an amino-ter- sponsible for binding to the signal sequence. minal signal sequence of 16–30 amino acids. Translation is temporarily halted until SRP54 Signal sequences are quite variable, but gener- interacts with the a-subunit of an SRP receptor ally have 6 to 12 hydrophobic amino acids that is embedded in the ER membrane through flanked by one or more positively charged resi- its b-subunit. Elongation arrest was first noted dues. As protein synthesis begins, the signal in one of Walter and Blobel’s pioneering stud- sequence is recognized by a signal recognition ies, in which they analyzed secretory protein 2 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a012872 Downloaded from http://cshperspectives.cshlp.org/ on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press ER Protein Secretion synthesis using microsomal membranes in vitro After the ribosomal/nascent protein/SRP (Walter and Blobel 1981). Further analysis and complex has docked to the SRP receptor, GTP binding studies showed that the SRP9 and hydrolysis releases SRP for another round of SRP14 proteins bind to the Alu domain (a re- nascent polypeptide recruitment. Structural de- petitive element) of the SRP RNA as a hetero- tails of the interaction between the eukaryotic dimer and are required for elongation arrest SRP, the SRP receptor, and the signal sequence (Siegel and Walter 1986; Strub et al. 1991). Par- are still awaited, but several structures of bac- ticles reconstituted with a carboxy-terminal terial homologs involved in the translocation truncation of SRP14 are unable to arrest prop- of polypeptides across their plasma membrane erly, despite having normal targeting and ribo- have been solved. For example, the heterodi- some-binding capabilities (Thomas et al. 1997; meric structure of the GTPase domains of Ffh Mason et al. 2000). The observation that chang- (a bacterial SRP54 homolog) and FtsY (a bacte- es in SRP RNA tertiary structure occurred upon rial SRP receptor a-subunit homolog) is stabi- binding to truncated SRP9/14 led to the pro- lized by the binding of two GTP molecules. This posal that RNA structural features were involved structure reveals how allosteric activation of the in mediating translation arrest (Thomas et al. two proteins is coordinated (Focia et al. 2004). 1997). A positively charged patch of predomi- More recently, a full-length Ffh:FtsY complex in nantly lysine residues was shown subsequently the pre-GTP hydrolysis state (cocrystallized to be essential for mediating the interaction be- with the GTPanalog GMPPCP) has been solved tween SRP14 and the SRP RNA (Mary et al. to 3.9 A˚ (Ataide et al. 2011). This work suggests 2010). An impressive cryo-electron microscopy that a large-scale repositioning event occurs to study of a mammalian SRP bound to a ribo- hand over the signal sequence from the M (me- some with a signal sequence has revealed the thionine-rich) domain of SRP54 (Ffh) to the arrangement of these components in an arrest- translocon. It has proved difficult to obtain ed state. In this structure, the Alu domain of the structures of SRP54 in combination with a sig- SRP RNA is positioned in the elongation-fac- nal sequence, but an elegant approach using an tor-binding site to bring about translational ar- Ffh-signal sequence fusion protein has allowed rest by direct competition (Halic et al. 2004). a 3.5 A˚ crystal structure to be determined from Why translational arrest should be need- dimers of the fusion protein (Janda et al. 2010). ed during secretory protein synthesis has been The structure is consistent with previous bio- food for thought for many years. In 2008, chemical experiments, showing that the signal Strub’s group showed that the amount of SRP peptide interacts with the M domain of Ffh and receptor at the ER membrane was a major lim- that hydrophobic residues in the core are nec- iting factor in determining translation rates essary for binding. Further studies using a range (Lakkaraju et al. 2008). Thus translational paus- of different signal peptides should help explain ing is necessary because it helps to keep nascent how Ffh, and ultimately SRP54, can maintain proteins translocation competent, until an SRP selectivity while tolerating diversity in signal receptor becomes available for docking, hence peptide length and sequence. preventing secretory-pathway proteins from be- The docking of SRP to the SRP receptor ing synthesized in the wrong compartment. positions the 60S subunit of the ribosome Further studies will be needed to determine above the translocon. The translocon is a pro- exactly how nascent chain length influences teinaceous channel that translocates the na- targeting, to explain the molecular sequence scent polypeptide chain into the ER lumen, of events that lead to resumption of translation and in mammals the translocon is composed upon SRP receptor engagement, and to explain of three principal subunits—Sec61a, b, and g. how translationally paused transcripts avoid The translocon is gated on the luminal side by mRNA degradation by RNA quality-control the ER chaperone BiP (grp78), which helps to mechanisms present in the cytosol (Becker preserve the barrier function of the ER mem- et al.
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