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 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.
Fig1. Overview of major protein-sorting pathways in eukaryotes. All nuclear DNA–encoded mRNAs are translated on cytosolic ribosomes. Right (nonsecretory pathways): Synthesis of proteins lacking an ER signal sequence is completed on free ribosomes (step 1). Those proteins that contain no targeting sequence are released into the cytosol and remain there (step 2). Proteins with an organelle-specific targeting sequence (pink) are first released into the cytosol (step 2 ) but are then imported into mitochondria, chloroplasts, peroxisomes, or the nucleus (steps 3–6). Mitochondrial and chloroplast proteins typically pass through the outer and inner membranes to enter the matrix or stromal space, respectively. Other proteins are sorted to other subcompartments of these organelles by additional sorting steps. Nuclear proteins enter and exit through visible pores in the nuclear envelope. Left (secretory pathway): Ribosomes synthesizing nascent proteins in the secretory pathway are directed to the rough endoplasmic reticulum (ER) by an ER signal sequence (pink; steps 1 and 2). After translation is completed on the ER, these proteins can move via transport vesicles to the Golgi complex (step 3). Further sorting delivers proteins either to the plasma membrane or to lysosomes (step 4a or 4b). The vesicle-based processes underlying the secretory pathway (steps 3 and 4, shaded box) are discussed in Chapter 14.
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 hydro phobic, 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.
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 2).
Fig2. Translation and translocation occur simultaneously. Cell-free experiments demonstrate that translocation of secretory proteins into microsomes is coupled to translation. Treatment of microsomes with EDTA, which chelates Mg2+ ions, strips them of associated ribosomes, allowing isolation of ribosome-free microsomes, which are equivalent to ER membranes. Protein synthesis is carried out in a cell-free system containing functional ribosomes, tRNAs, ATP, GTP, 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 (resulting in a decrease in molecular weight) only if microsomes are present during protein synthesis (b).
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. 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.
