Translocation of Secretory Proteins Across the ER Membrane

All eukaryotic cells use essentially the same secretory pathway for synthesizing and sorting secreted proteins and soluble luminal proteins in the ER, Golgi, and lysosomes (see Figure 16-1, left). For simplicity, we refer to these proteins collectively as secretory proteins. Although all cells secrete a variety of proteins (e.g., extracellular matrix proteins), certain types of cells are specialized for secretion of large

Cytosol ER lumen ER membrane

Cytosol ER lumen ER membrane

Free ribosomes

Attached ribosomes

Free ribosomes

Attached ribosomes

▲ FIGURE 16-2 Electron micrograph of ribosomes attached to the rough ER in a pancreatic acinar cell. Most of the proteins synthesized by this type of cell are to be secreted and are formed on membrane-attached ribosomes. A few membrane-unattached (free) ribosomes are evident; presumably, these are synthesizing cytosolic or other nonsecretory proteins. [Courtesy of G. Palade.]

mRNA

Labeled secretory protein

Rough ER

Microsomes with attached ribosomes mRNA

Labeled secretory protein

Rough ER

Microsomes with attached ribosomes

( protease

Digestion of secretory protein

No digestion of secretory protein

( protease

Digestion of secretory protein

No digestion of secretory protein amounts of specific proteins. Pancreatic acinar cells, for instance, synthesize large quantities of several digestive enzymes that are secreted into ductules that lead to the intestine. Because such secretory cells contain the organelles of the secretory pathway (e.g., ER and Golgi) in great abundance, they have been widely used in studying this pathway.

Early pulse-labeling experiments with pancreatic acinar cells showed that radioactively labeled amino acids are incorporated primarily into newly synthesized secretory proteins. The ribosomes synthesizing these proteins are actually bound to the surface of the ER. As a consequence, the portion of the ER that receives proteins entering the secretory pathway is known as the rough ER because these membranes are densely studded with ribosomes (Figure 16-2). When cells are homogenized, the rough ER breaks up into small closed vesicles, termed rough microsomes, with the same orientation (ribosomes on the outside) as that found in the intact cell. The experiments depicted in Figure 16-3, in

EXPERIMENTAL FIGURE 16-3 Labeling experiments demonstrate that secretory proteins are localized to the ER lumen shortly after synthesis. Cells are incubated for a brief time with radiolabeled amino acids, so that only newly synthesized proteins become labeled. The cells then are homogenized, fracturing the plasma membrane and shearing the rough ER into small vesicles called microsomes. Because they have bound ribosomes, microsomes have a much greater buoyant density than other membranous organelles and can be separated from them by a combination of differential and sucrose density-gradient centrifugation (Chapter 5). The purified microsomes are treated with a protease in the presence or absence of a detergent. The labeled secretory proteins associated with the microsomes are digested by added proteases only if the permeability barrier of the microsomal membrane is first destroyed by treatment with detergent. This finding indicates that the newly made proteins are inside the microsomes, equivalent to the lumen of the rough ER.

which microsomes isolated from pulse-labeled cells are treated with a protease, demonstrate that although secretory proteins are synthesized on ribosomes bound to the cytosolic face of the ER membrane, they become localized in the lumen of ER vesicles during their synthesis.

A Hydrophobic N-Terminal Signal Sequence Targets Nascent Secretory Proteins to the ER

After synthesis of a secretory protein begins on free ribo-somes in the cytosol, a 16- to 30-residue ER signal sequence in the nascent protein directs the ribosome to the ER membrane and initiates translocation of the growing polypeptide across the ER membrane (see Figure 16-1, left). An ER signal sequence typically is located at the N-terminus of the protein, the first part of the protein to be synthesized. The signal sequences of different secretory proteins contain one or more positively charged amino acids adjacent to a continuous stretch of 6-12 hydrophobic residues (the core), but otherwise they have little in common. For most secretory proteins, the signal sequence is cleaved from the protein while it is still growing on the ribosome; thus, signal sequences are usually not present in the "mature" proteins found in cells.

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 introduction of charged amino acids into the hy-drophobic core by mutation, can abolish the ability of the N-terminus of a protein to function as a signal sequence. As a consequence, the modified protein remains in the cytosol, unable to cross the ER membrane into the lumen. Using recombinant DNA techniques, researchers have produced cy-tosolic proteins with added N-terminal amino acid sequences. Provided the added sequence is sufficiently long and hydrophobic, such a modified cytosolic protein is translocated to the ER lumen. Thus the hydrophobic residues

(a) Cell-free protein synthesis; no microsomes present

Add microsome membranes

(a) Cell-free protein synthesis; no microsomes present

Add microsome membranes

N-terminal signal sequence

Completed proteins with signal sequences

N-terminal signal sequence

Completed proteins with signal sequences

No incorporation into microsomes; no removal of signal sequence

(b) Cell-free protein synthesis; microsomes present

Cotranslational transport of protein into microsome and removal of signal sequence

Mature protein chain without signal sequence in the core of ER signal sequences form a binding site that is critical for the interaction of signal sequences with receptor proteins on the ER membrane.

Biochemical studies utilizing a cell-free protein-synthesizing system, mRNA encoding a secretory protein, and micro-somes stripped of their own bound ribosomes have clarified the function and fate of ER signal sequences. Initial experiments with this system demonstrated that a typical secretory protein is incorporated into microsomes and has its signal sequence removed only if the microsomes are present during protein synthesis (Figure 16-4). Subsequent experiments were designed to determine the precise stage of protein synthesis at which microsomes must be present in order for translocation to occur. In these experiments, a drug that prevents initiation of translation was added to protein-synthesizing reactions at different times after protein synthesis had begun, and then stripped microsomes were added to the reaction mixtures. These experiments showed that microsomes must be added before the first 70 or so amino acids are linked together in order for the completed secretory protein to be localized in the microsomal lumen. At this point, the first 40 amino acids or so protrude from the ribosome, including the signal sequence that later will be cleaved off, and the next 30 or so amino acids are still buried within a channel in the ribosome. Thus the transport of most secretory proteins into the ER lumen occurs while the nascent protein is still bound to the ribosome and being elongated, a process referred to as cotranslational translocation.

M EXPERIMENTAL FIGURE 16-4 Cell-free experiments demonstrate that translocation of secretory proteins into microsomes is coupled to translation. Treatment of microsomes with EDTA, which chelates Mg+2 ions, strips them of associated ribosomes, allowing isolation of ribosome-free microsomes, which are equivalent to ER membranes (see Figure 16-3). Synthesis is carried out in a cell-free system containing functional ribosomes, tRNAs, ATP; GTR and cytosolic enzymes to which mRNA encoding a secretory protein is added. The secretory protein is synthesized in the absence of microsomes (a), but is translocated across the vesicle membrane and loses its signal sequence only if microsomes are present during protein synthesis (b).

Cotranslational Translocation Is Initiated by Two GTP-Hydrolyzing Proteins

Since secretory proteins are synthesized in association with the ER membrane but not with any other cellular membrane, a signal-sequence recognition mechanism must target them there. The two key components in this targeting are the signal-recognition particle (SRP) and its receptor located in the ER membrane. The SRP is a cytosolic ribonucleoprotein particle that transiently binds simultaneously to the ER signal sequence in a nascent protein, to the large ribosomal unit, and to the SRP receptor.

Six discrete polypeptides and a 300-nucleotide RNA compose the SRP (Figure 16-5a). One of the SRP proteins (P54) can be chemically cross-linked to ER signal sequences, evidence that this particular protein is the subunit that binds to the signal sequence in a nascent secretory protein. A region of P54 containing many amino acid residues with hy-drophobic side chains is homologous to a bacterial protein known as Ffh, which performs an analogous function to P54 in the translocation of proteins across the inner membrane of bacterial cells. The structure of Ffh contains a cleft whose inner surface is lined by hydrophobic side chains (Figure 16-5b). The hydrophobic region of P54 is thought to contain an analogous cleft that interacts with the hydrophobic N-termini of nascent secretory proteins and selectively targets them to the ER membrane. Two of the SRP proteins, P9 and P14, interact with the ribosome, while P68 and P72 are required for protein translocation.

In the cell-free translation system described previously, the presence of SRP slows elongation of a secretory protein when microsomes are absent, thereby inhibiting synthesis of the complete protein (see Figure 16-4). This finding suggests that interaction of the SRP with both the nascent chain of a secretory protein and with the free ribosome prevents the nascent chain from becoming too long for translocation into the ER. Only after the SRP/nascent chain/ribosome complex has bound to the SRP receptor in the ER membrane does SRP release the nascent chain, allowing elongation at the normal rate.

Figure 16-6 summarizes our current understanding of secretory protein synthesis and the role of the SRP and its

(a) Signal-recognition particle (SRP) P54

Binds ER signal sequence

Required for protein translocation

P9/P14

Interact with ribosomes

(b) Ffh signal sequence-binding domain

(b) Ffh signal sequence-binding domain

receptor in this process. The SRP receptor is an integral membrane protein made up of two subunits: an a subunit and a smaller p subunit. Treatment of microsomes with very small amounts of protease cleaves the a subunit very near its site of attachment to the membrane, releasing a soluble form of the SRP receptor. Protease-treated microsomes are unable to bind the SRP/nascent chain/ribosome complex or support cotranslational translocation. The soluble SRP receptor fragment, however, retains its ability to interact with the SRP/nascent chain/ribosome complex, causing release of SRP and allowing chain elongation to proceed. Thus the SRP and SRP receptor not only help mediate interaction of a

FIGURE 16-5 Structure of the signal-recognition particle (SRP). (a) The SRP comprises one 300-nucleotide RNA and six proteins designated P9, P14, P19, P54, P68, and P72. (The numeral indicates the molecular weight X 103.) All proteins except P54 bind directly to the RNA. (b) The bacterial Ffh protein is homologous to the portion of P54 that binds ER signal sequences. This surface model shows the binding domain in Ffh, which contains a large cleft lined with hydrophobic amino acids (purple) whose side chains interact with signal sequences. [Part (a) see K. Strub et al., 1991, Mol. Cell Biol. 11:3949; and S. High and B. Dobberstein, 1991, J. Cell Biol. 113:229. Part (b) adapted from R. J. Keenan et al., 1998, Cell 94:181.]

nascent secretory protein with the ER membrane but also act together to permit elongation and synthesis of complete proteins only when ER membranes are present.

Ultimately, the SRP and SRP receptor function to bring ribosomes that are synthesizing secretory proteins to the ER membrane. The coupling of GTP hydrolysis to this targeting process is thought to contribute to the fidelity by which signal sequences are recognized. Probably the energy from GTP hydrolysis is used to release proteins lacking proper signal sequences from the SRP and SRP receptor complex, thereby preventing their mistargeting to the ER membrane. (A similar coupling of GTP hydrolysis with binding of translation elongation factors to ribosomes increases the fidelity of translation by ejecting aminoacyl-tRNA molecules that cannot form correct base pairs with the codons in mRNA.) Interaction of the SRP/nascent chain/ribosome complex with the SRP receptor is promoted when GTP is bound by both the P54 subunit of SRP and the a subunit of the SRP receptor (see Figure 16-6). Subsequent transfer of the nascent chain and ribosome to a site on the ER membrane where translocation can take place allows hydrolysis of the bound GTP. After dissociating, SRP and its receptor release the bound GDP and recycle to the cytosol ready to initiate another round of interaction between ribosomes synthesizing nascent secretory proteins with the ER membrane.

Passage of Growing Polypeptides Through the Translocon Is Driven by Energy Released During Translation

Once the SRP and its receptor have targeted a ribosome synthesizing a secretory protein to the ER membrane, the ribo-some and nascent chain are rapidly transferred to the translocon, a protein-lined channel within the membrane. As translation continues, the elongating chain passes directly from the large ribosomal subunit into the central pore of the translocon. The 60S ribosomal subunit is aligned with the pore of the translocon in such a way that the growing chain is never exposed to the cytoplasm and does not fold until it reaches the ER lumen (see Figure 16-6).

The translocon was first identified by mutations in the yeast gene encoding Sec61a, which caused a block in the

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