Protein Targeting and Degradation:- Glycosylation Plays a Key Role in Protein Targeting
In the ER lumen, newly synthesized proteins are further modified in several ways. Following the removal of signal sequences, polypeptides are folded, disulfide bonds formed, and many proteins glycosylated to form glycoproteins. In many glycoproteins the linkage to their oligosaccharides is through Asn residues. These N linked oligosaccharides are diverse, but the pathways by which they form have a common first step. A 14-residue core oligosaccharide is built up in a step wise fashion, then transferred from a dolichol phosphate donor molecule to certain Asn residues in the protein (Fig. 1). The transferase is on the lumenal face of the ER and thus cannot catalyze glycosylation of cytosolic proteins. After transfer, the core oligosaccharide is trimmed and elaborated in different ways on different proteins, but all N-linked oligosaccharides retain a pen tasaccharide core derived from the original 14 residue oligosaccharide. Several antibiotics act by interfering with one or more steps in this process and have aided in elucidating the steps of protein glycosylation. The best-characterized is tunicamycin, which mimics the structure of UDP-N-acetylglucosamine and blocks the first step of the process (Fig. 1, step 1). A few proteins are O-glycosylated in the ER, but most O-glycosylation occurs in the Golgi complex or in the cytosol (for proteins that do not enter the ER). Suitably modified proteins can now be moved to a variety of intracellular destinations. Proteins travel from the ER to the Golgi complex in transport vesicles (Fig. 2). In the Golgi complex, oligosaccharides are O linked to some proteins, and N-linked oligosaccharides are further modified. By mechanisms not yet fully understood, the Golgi complex also sorts proteins and sends them to their final destinations. The processes that segregate proteins targeted for secretion from those targeted for the plasma membrane or lysosomes must distinguish among these proteins on the basis of structural features other than signal sequences, which were removed in the ER lumen.
FIGURE 1 Synthesis of the core oligosaccharide of glycoproteins. The core oligosaccharide is built up by the successive addition of monosaccharide units. 1 , 2 The first steps occur on the cytosolic face of the ER. 3 Translocation moves the incomplete oligosac charide across the membrane (mechanism not shown), and 4 completion of the core oligosaccharide occurs within the lumen of the ER. The precursors that contribute additional mannose and glucose residues to the growing oligosaccharide in the lumen are dolichol phosphate derivatives. In the first step in the construction of the N linked oligosaccharide moiety of a glycoprotein, 5, 6 the core oligosaccharide is transferred from dolichol phosphate to an Asn residue of the protein within the ER lumen. The core oligosaccharide is then further modified in the ER and the Golgi complex in pathways that differ for different proteins. The five sugar residues shown sur rounded by a beige screen (after step 7) are retained in the final structure of all N-linked oligosaccharides. 8 The released dolichol pyrophosphate is again translocated so that the pyrophosphate is on the cytosolic face of the ER, then 9 a phosphate is hydrolytically re moved to regenerate dolichol phosphate.
FIGURE 2 Pathway taken by proteins destined for lysosomes, the plasma membrane, or secretion. Proteins are moved from the ER to the cis side of the Golgi complex in transport vesicles. Sorting occurs primarily in the trans side of the Golgi complex.
This sorting process is best understood in the case of hydrolases destined for transport to lysosomes. On arrival of a hydrolase (a glycoprotein) in the Golgi com plex, an as yet undetermined feature (sometimes called a signal patch) of the three-dimensional structure of the hydrolase is recognized by a phosphotransferase, which phosphorylates certain mannose residues in the oligo saccharide . The presence of one or more mannose 6-phosphate residues in its N-linked oligosac charide is the structural signal that targets the protein to lysosomes. A receptor protein in the membrane of the Golgi complex recognizes the mannose 6-phosphate signal and binds the hydrolase so marked. Vesicles containing these receptor-hydrolase complexes bud from the trans side of the Golgi complex and make their way to sorting vesicles. Here, the receptor-hydrolase com plex dissociates in a process facilitated by the lower pH in the vesicle and by phosphatase-catalyzed removal of phosphate groups from the mannose 6-phosphate residues. The receptor is then recycled to the Golgi com plex, and vesicles containing the hydrolases bud from the sorting vesicles and move to the lysosomes. In cells treated with tunicamycin , hydro lases that should be targeted for lysosomes are instead secreted, confirming that the N-linked oligosaccharide plays a key role in targeting these enzymes to lysosomes. The pathways that target proteins to mitochondria and chloroplasts also rely on amino-terminal signal sequences. Although mitochondria and chloroplasts contain DNA, most of their proteins are encoded by nuclear DNA and must be targeted to the appropriate organelle. Unlike other targeting pathways, however, the mitochondrial and chloroplast pathways begin only after a precursor protein has been completely synthesized and released from the ribosome. Precursor proteins destined for mitochondria or chloroplasts are bound by cytosolic chaperone proteins and delivered to receptors on the exterior surface of the target organelle. Specialized translocation mechanisms then transport the protein to its final destination in the organelle, after which the signal sequence is removed.
