Protein Targeting and Degradation:- Posttranslational Modification of Many Eukaryotic Proteins Begins in the Endoplasmic Reticulum
Perhaps the best-characterized targeting system begins in the ER. Most lysosomal, membrane, or secreted proteins have an amino-terminal signal sequence (Fig. 1) that marks them for translocation into the lumen of the ER; hundreds of such signal sequences have been determined. The carboxyl terminus of the signal sequence is defined by a cleavage site, where protease action removes the sequence after the protein is imported into the ER. Signal sequences vary in length from 13 to 36 amino acid residues, but all have the following features: (1) about 10 to 15 hydrophobic amino acid residues; (2) one or more positively charged residues, usually near the amino terminus, preceding the hydrophobic sequence; and (3) a short sequence at the carboxyl terminus (near the cleavage site) that is relatively polar, typically having amino acid residues with short side chains (especially Ala) at the positions closest to the cleavage site. As originally demonstrated by George Palade, proteins with these signal sequences are synthesized on ribosomes attached to the ER. The signal sequence itself helps to direct the ribosome to the ER, as illustrated by steps 1 through 8 in Figure 2. 1 The targeting pathway begins with initiation of protein synthesis on free ribosomes. 2 The signal sequence appears early in the synthetic process, because it is at the amino terminus, which as we have seen is synthesized first. 3 As it emerges from the ribosome, the signal sequence—and the ribosome itself—are bound by the large signal recognition particle (SRP); SRP then binds GTP and halts elongation of the polypeptide when it is about 70 amino acids long and the signal sequence has completely emerged from the ribosome. 4 The GTP-bound SRP now directs the ribosome (still bound to the mRNA) and the incomplete polypeptide to GTP-bound SRP receptors in the cytosolic face of the ER; the nascent polypep tide is delivered to a peptide translocation complex in the ER, which may interact directly with the ribosome. 5 SRP dissociates from the ribosome, accompanied by hydrolysis of GTP in both SRP and the SRP receptor. 6 Elongation of the polypeptide now resumes, with the ATP-driven translocation complex feeding the growing polypeptide into the ER lumen until the complete protein has been synthesized. 7 The signal sequence is removed by a signal peptidase within the ER lumen; 8 the ribosome dissociates and is recycled.
FIGURE 1 Translocation into the ER directed by amino-terminal signal sequences of some eukaryotic proteins. The hydrophobic core (yellow) is preceded by one or more basic residues (blue). Note the polar and short-side-chain residues immediately preceding (to the left of, as shown here) the cleavage sites (indicated by red arrows).
FIGURE 2 Directing eukaryotic proteins with the appropriate signals to the endoplasmic reticulum. This process involves the SRP cycle and translocation and cleavage of the nascent polypeptide. The steps are described in the text. SRP is a rod-shaped complex containing a 300 nucleotide RNA (7SL-RNA) and six different proteins (combined Mr 325,000). One protein subunit of SRP binds directly to the signal sequence, inhibiting elongation by sterically blocking the entry of aminoacyl-tRNAs and inhibiting peptidyl transferase. Another protein subunit binds and hydrolyzes GTP. The SRP receptor is a heterodimer of (Mr 69,000) and (Mr 30,000) subunits, both of which bind and hydrolyze multiple GTP molecules during this process.
