Section 17.

4Translocation of Secretory Proteins across the ER Membrane

In the next several sections, we examine in detail the events occurring in the secretory pathway (see Figure 17-13). We begin in this section by focusing on three key questions: How are certain proteins targeted to the secretory pathway? How do secretory proteins actually cross the membrane? And what is the source of energy for the transport of proteins across the ER membrane?

A Signal Sequence on Nascent Secretory Proteins Targets Them to the ER and Is Then Cleaved Off
Go to: Top As noted earlier, synthesis of most secretory proteins begins on free ribosomes in the cytosol. The presence of a 16to 30-residue ER signal sequence directs the ribosome to the ER membrane and initiates transport of the growing poly-peptide across the ER membrane. An ER signal sequence typically is located at the N-terminus of the protein and contains one or more positively charged amino acids followed by a continuous stretch of 6 12 hydrophobic residues (Table 17-4); except for these features, the signal sequences of different secretory proteins have little in common. Signal sequences are not normally found on complete polypeptides made in cells, implying that the signal sequence is cleaved from the protein while it is still growing on the ribosome.

Table 17-4 Amino Acid Sequences of ER Signal Peptides in Three Eukaryotic Proteins. The hydrophobic core of ER signal sequences is essential for their function. For instance, the specific deletion of several of the hydrophobic amino acids from a signal sequence, or the mutation of one of them to a charged amino acid, abolishes the ability of the protein to cross the ER membrane into the lumen. Other experiments indicate that addition (by recombinant DNA techniques) of any random N-terminal amino acid sequence, provided it is sufficiently long and hydrophobic, will cause a normally cytosolic protein to be translocated to the ER lumen. These hydrophobic residues form a binding site that is critical for the interaction of signal sequences with receptor proteins on the ER membrane. Experiments with microsomes have clarified the function and fate of ER signal sequences. When the mRNA encoding a secretory protein is translated in a cell-free system containing ribosomes, tRNAs, ATP and GTP, and cytosolic enzymes but with no ER membranes, the protein, with its signal sequence attached, is released into the cytosol. If microsomes that have been stripped of their own ribosomes are then added to the reaction mixture, the protein generally is not incorporated into the ER lumen and its signal sequence remains attached (Figure 17-15a). However, if these membranes are present during the cell-free synthesis of a secretory protein, the protein is found in the ER lumen with the signal sequence removed (Figure 17-15b). Thus, a newly made secretory protein can cross the ER membrane and have its signal sequence removed only if the membrane is present during protein synthesis. The enzyme signal peptidase, which normally cleaves off the signal sequences, is localized to the ER lumen.

Figure 17-15

The cotranslational insertion of secretory proteins into microsomes, which are formed by shearing of the ER into small vesicles. Treatment of microsomes with EDTA, which chelates Mg2+ ions, strips them of associated ribosomes, leaving (more...) In subsequent experiments, microsomes were added at different times after the synchronized cell-free synthesis of a secretory protein began. In order for the subsequently completed secretory protein to be localized in the ER lumen, microsomes must be added before the first 70 or so amino acids are polymerized. At this point, about 40 amino acids protrude from the ribosomes, including the signal sequence that later will be cleaved off, and about 30 amino acids are buried in a channel or tunnel in the ribosome. Thus the transport of most secretory proteins into the ER lumen particularly those with more than 100 amino acids must occur during translation, a process referred to as cotranslational transport. Some small secretory proteins, such as the yeast mating factor (70 amino acids), exhibit post-translational transport into the ER lumen. Such proteins are synthesized in their entirety on free cytosolic ribosomes and then released into the cytosol. Here they are bound by chaperones, which keep them in an unfolded state, and subsequently are translocated across the ER membrane. Thus the targeting of these proteins to the ER lumen is similar to targeting of proteins to the mitochondrial matrix or chloroplast stroma.

Two Proteins Initiate the Interaction of Signal Sequences with the ER Membrane
Go to: Top Since secretory proteins are synthesized in association with the ER membrane but not with any other cellular membrane, some signal-sequence recognition mechanism must target them there. As depicted in Figure 17-16, the two key components in this targeting are the signal-recognition particle (SRP) and the SRP receptor. SRP is a cytosolic particle that transiently binds to the ER signal sequence in a nascent protein, to the large ribosomal unit, and to the SRP receptor in the ER membrane.

Figure 17-16 Synthesis of secretory proteins on the rough ER. Synthesis begins on an unattached ribosome in the cytosol. The complete N-terminal signal sequence emerges from the ribosome only when the polypeptide is about 70 amino acids long, because (more...) A simple experiment provided the first clue to the existence of the SRP. When microsomes, stripped of their ribosomes, were exposed to a solution of 0.5 M NaCl and then recovered by centrifugation, the treated microsomes were unable to support the transmembrane movement of newly synthesized secretory proteins in a cell-free system. However, when the proteins removed by the NaCl treatment were returned to the preparation, secretory proteins were incorporated into the lumen of the vesicles as in Figure 17-15b. A single active component now called the signal-recognition particle (SRP) subsequently was purified from the mixture of proteins extracted by NaCl. The SRP is a ribonucleoprotein composed of six discrete polypeptides and a 300-nucleotide RNA (Figure 17-17).

Figure 17-17

Structure of the signal-recognition particle (SRP). SRP comprises one 300-nucleotide RNA and six proteins designated P9, P14, P19, P54, P68, and P72. (The numeral indicates the molecular weight 10 3). All proteins except (more...) One of the SRP proteins (P54) can be chemically cross-linked to ER signal sequences, evidence that this 54,000MW protein is the one that binds to the signal sequence in a nascent secretory protein. One region of P54 contains a large number of clustered methionine residues whose hydrophobic side chains are thought to protrude outward and bind to the hydrophobic side chains that form the core of an ER signal sequence. Other SRP proteins, P9 and P14, interact with the ribosome, while P68 and P72 are required for protein translocation. Experiments in the wheat-germ cell-free system (see Figure 17-15) have shown that SRP arrests secretory-protein synthesis after polymerization of about 70 amino acids when microsomes are absent. Thus SRP not only helps mediate interaction of a nascent protein with the ER membrane but also prevents synthesis of a complete protein in the absence of rough ER membranes. The complex of a SRP, nascent chain, and ribosome binds to the SRP receptor in the ER membrane (see Figure 1716). This receptor contains two polypeptide subunits: a transmembrane subunit of about 300 amino acids, and a peripheral subunit of about 640 amino acids. The treatment of ER membranes with minute amounts of protease cleaves the subunit very near its site of attachment to the membrane, releasing a soluble form of the SRP receptor. This soluble fragment can relieve the block in chain elongation that is imposed by SRP in cell-free systems, evidence that the SRP receptor binds to the SRP and possibly also to the ribosome.

Polypeptides Move through the Translocon into the ER Lumen

Go to: Top SRP and its receptor only initiate the transfer of the nascent chain across the ER membrane; then they dissociate from the chain, which is transferred to a set of transmembrane proteins, collectively called the translocon (see Figure 17-16). Once the nascent chaintranslocon complex is assembled, the elongating chain passes directly from the large ribosomal subunit into the centers of the translocon, a protein-lined channel within the membrane. The growing chain is never exposed to the cytosol and does not fold until it reaches the ER lumen. Thus the protein crosses the membrane in an unfolded state; if newly made secretory proteins were allowed to fold in the cytosol, they probably would be unable to cross the ER membrane. The experiment described in Figure 17-18 identified two abundant ER membrane proteins that can be chemically cross-linked to a nascent chain, indicating that they form part of the mammalian translocon. One of these proteins, named TRAM (translocating chainassociated membrane) protein, can be chemically cross-linked to the ER signal sequence; this protein may bind the signal sequence as it is handed over by the SRP to the translocon. TRAM protein, which spans the ER membrane at least eight times, is essential for protein translocation. The second mammalian protein identified in this cross-linking experiment, Sec61p, is similar in amino acid sequence to the protein encoded by the yeast sec61 gene. Analysis of yeast sec61 mutants show they are class A mutants, defective in the translocation of secretory proteins into the ER lumen (see Figure 17-14), further evidence that the homologous mammalian protein, Sec61p, has a similar function. Mammalian Sec61p, which has 10 membrane-spanning helices, is tightly bound to two other proteins, termed Sec61 and Sec61; the three polypeptides form the Sec61 complex. This protein complex, which also binds tightly to ribosomes, may function to attach the 60S subunit of ribosomes that are synthesizing secretory proteins to the ER membrane.

Figure 17-18 Identification of the proteins that form the translocon, the transmembrane channel through which a nascent secretory protein passes into the ER lumen. An mRNA was synthesized that encoded the first 70 amino acids of the secretory protein (more...)

Subsequent biochemical experiments showed that phospholipid vesicles reconstituted in vitro and containing only the SRP receptor, TRAM, and the Sec61 complex are functional in translocating nascent secretory proteins. Thus these are the only mammalian ER-membrane proteins required for translocation. The actual channel is lined by three or four Sec61 complexes. Images of the channel have been generated by computer averaging of serial reconstructions of electron micrographs of freeze-fractured purified Sec61p channels reconstituted in liposomes. These show the channel as a roughly pentagonal cylinder, 56 nm high and 8.5 nm in diameter, with a central pore, 2 nm in diameter, that extends throughout the protein complex perpendicular to the plane of the membrane (Figure 17-19).

Figure 17-19 Electron microscopic view of a translocon channel. Purified Sec61p translocon subunits were solubilized in detergents; ribosomes were added and the preparation was reconstituted into phospholipid bilayers (see Figure 154). The preparation was (more...) In the absence of attached ribosomes and protein translocation, the translocon channel is closed at the cytosolic end by a segment of the Sec61p protein. Binding of a ribosome-nascent chain complex causes this gate to open and then form a tight seal between the ribosome and translocon such that small molecules cannot pass though the translocon pore. Opening of the gate allows a loop of the nascent chain, containing the signal sequence and 30 adjacent amino acids to insert into the translocon pore (see Figure 17-16). After cleavage of the signal sequence, the growing polypeptide moves through the pore into the ER lumen.

GTP Hydrolysis Powers Protein Transport into the ER in Mammalian Cells

Go to: Top As we learned earlier in this chapter, the uptake of proteins into mitochondria and chloroplasts requires energy. Likewise, energy is required for insertion of the signal sequence into the translocon and for the subsequent translocation of the growing polypeptide chain across the ER membrane. As Figure 17-20 shows, release of GDP and binding of GTP, by the P54 subunit of SRP and by the subunit of the SRP receptor, promote formation of the complex comprising a ribosome, nascent chain, SRP, and SRP receptor. Subsequent hydrolysis of the bound GTP promotes dissociation of this complex and formation of the nascent chain translocon complex. After dissociating, SRP and its receptor recycle to initiate the interaction of additional nascent chains with the ER membrane.

Figure 17-20 Cycles of GDP-GTP exchange and GTP hydrolysis that drive insertion of nascent secretory proteins into the translocon. Both the P54 subunit of SRP and the subunit of the SRP receptor bind and hydrolyze GTP. In step 1, SRP containing (more...) In yeast, a homolog of the chaperone Hsc70 is localized to the ER lumen and binds nascent chains as they pass through the translocon (see Figure 17-16). Studies with yeasts expressing mutant forms of this Hsc70 protein have shown that ATP hydrolysis by Hsc70 is essential for cotranslational transport of proteins across the ER membrane. ATP hydrolysis by this luminal Hsc70 chaperone also powers the post-translational uptake of the small yeast factor into the ER lumen. (Recall that small secretory proteins, such as factor, are synthesized in the cytosol and then imported.) However, in mammalian cells cotranslational translocation does not require ATP hydrolysis. Instead, hydrolysis of GTP during protein synthesis itself is thought to drive the nascent polypeptide from the membrane-attached ribosome across the ER membrane.

Table 17-4Amino Acid Sequences of ER Signal Peptides in Three Eukaryotic Proteins

Protein Preproalbumin Pre-IgG light chain Prelysozyme Amino Acid Sequence* Met-Lys-Trp-Val-Thr-Phe-Leu-Leu-Leu-Leu-Phe-Ile-Ser-Gly-Ser-Ala-Phe-Ser Arg . . . Met-Asp-Met-Arg-Ala-Pro-Ala-Gln-Ile-Phe-Gly-Phe-Leu-Leu-Leu-Leu-Phe-Pro-Gly- ThrArg-Cys Asp . . . Met-Arg-Ser-Leu-Leu-Ile-Leu-Val-Leu-Cys-Phe-Leu-Pro-Leu-Ala-Ala-Leu-Gly Lys . . .

Figure 17-15The cotranslational insertion of secretory proteins into microsomes, which are formed by shearing of the ER into small vesicles

Figure 17-16Synthesis of secretory proteins on the rough ER

Figure 17-17Structure of the signal-recognition particle (SRP)

Figure 17-18Identification of the proteins that form the translocon, the transmembrane channel through which a nascent secretory protein passes into the ER lumen

Figure 17-20Cycles of GDP-GTP exchange and GTP hydrolysis that drive insertion of nascent secretory proteins into the translocon

SUMMARY
Go to: Top Secretory proteins, enzymes destined for the ER, Golgi complex, and lysosomes, and integral plasmamembrane proteins are synthesized on ribosomes bound to the rough ER. Synthesis of this class of proteins is initiated on membrane-unattached ribosomes. A sequence of hydrophobic amino acids, the ER signal sequence, is recognized and bound by a signalrecognition particle (SRP), which in turn is bound by an SRP receptor on the rough ER membrane. Generally, ER signal sequences are located at the N-terminus and are cleaved from the protein in the rough ER lumen. The SRP directs both the binding of the ribosome to the ER membrane and the insertion of the nascent protein into the transmembrane channel (see Figure 17-16). Hydrolysis of GTP by the P54 subunit of SRP and the subunit of the SRP receptor is essential for this process. Proteins cross the ER membrane in an unfolded state through a protein-lined channel, the translocon, a multiprotein complex composed of TRAM protein and the Sec61 complex. In mammalian cells, the only integral ER proteins required for translocation of nascent secretory proteins are the SRP receptor, TRAM, and the Sec61 complex.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed. Copyright 2000, W. H. Freeman and Company.

Molecular Cell Biology. 4th edition. Lodish H, Berk A, Zipursky SL, et al. New York: W. H. Freeman; 2000.
[Table of Contents Page]

In this Page
A Signal Sequence on Nascent Secretory Proteins Targets Them to the ER and Is Then Cleaved Off Two Proteins Initiate the Interaction of Signal Sequences with the ER Membrane Polypeptides Move through the Translocon into the ER Lumen GTP Hydrolysis Powers Protein Transport into the ER in Mammalian Cells SUMMARY

Recent activity
ClearTurn Off

Molecular Cell Biology

Molecular Cell Biology

Bookshelf

Translocation of Secretory Proteins across the ER Membrane - Molecular Cell Biol...

Translocation of Secretory Proteins across the ER Membrane - Molecular Cell Biology

Bookshelf

See more...
You are here: NCBI > Literature > Bookshelf

Write to the Help Desk

imple NCBI Directory

GETTING STARTED
NCBI Education NCBI Help Manual NCBI Handbook Training & Tutorials

RESOURCES
Chemicals & Bioassays Data & Software DNA & RNA Domains & Structures Genes & Expression Genetics & Medicine Genomes & Maps Homology Literature Proteins Sequence Analysis Taxonomy Training & Tutorials Variation

POPULAR
PubMed Nucleotide BLAST PubMed Central Gene Bookshelf Protein OMIM Genome

SNP Structure

FEATURED
GenBank Reference Sequences Map Viewer Genome Projects Human Genome Mouse Genome Influenza Virus Primer-BLAST Sequence Read Archive

NCBI INFORMATION
About NCBI Research at NCBI NCBI Newsletter NCBI FTP Site NCBI on Facebook NCBI on Twitter NCBI on YouTube NLM

NIH

DHHS

USA.gov