After synthesis of a secretory protein begins on free ribosomes in the cytosol, a 16–30-residue ER targeting 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 13-1, left). An ER targeting sequence, typically located at the N-terminus of the protein, is usually known as a signal sequence. The signal sequences of different secretory proteins all contain one or more positively charged amino acids adjacent to a continuous stretch of 6–12 hydrophobic residues (known as the hydrophobic core), but otherwise have little in common. The signal sequence is cleaved from most secretory proteins while they are still elongating on the ribosome; thus signal sequences are usually not present in the mature proteins found in cells.

The hydrophobic core of an ER signal sequence is essential for its 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 hydrophobic 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. Conversely, signal sequences can be added to normally cytosolic proteins using recombinant DNA techniques. Provided the added sequence is sufficiently long and hydrophobic, such a modified cytosolic protein can acquire the ability to be translocated to the ER lumen. The hydrophobic residues in the core of an ER signal sequence form a binding site that is critical for the interaction of the signal sequence with the machinery responsible for targeting the protein to the ER membrane.

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Biochemical studies using a cell-free protein-synthesizing system, mRNA encoding a secretory protein, and microsomes stripped of their own bound ribosomes have elucidated 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. If microsomes are added to the system after protein synthesis is completed, no protein transport into the microsomes occurs (Figure 13-4). 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, microsomes were added to the reaction mixtures at different times after protein synthesis had begun. These experiments showed that microsomes must be added before the first 70 or so amino acids are translated in order for the completed secretory protein to be localized in the microsomal lumen. At this point, the first 40 or so amino acids protrude from the ribosome, including the signal sequence that will later be cleaved off, and the next 30 or so amino acids are still buried within a channel in the ribosome (see Figure 5-26). Thus the transport of most secretory proteins into the ER lumen begins while the incompletely synthesized (nascent) protein is still bound to the ribosome, a process referred to as cotranslational translocation.

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