The levels of proteins within cells are determined not only by rates of synthesis, but also by rates of degradation. The half-lives of proteins within cells vary widely, from minutes to several days, and differential rates of protein degradation are an important aspect of cell regulation. Many rapidly degraded proteins function as regulatory molecules, such as transcription factors. The rapid turnover of these proteins is necessary to allow their levels to change quickly in response to external stimuli. Other proteins are rapidly degraded in response to specific signals, providing another mechanism for the regulation of intracellular enzyme activity. In addition, faulty or damaged proteins are recognized and rapidly degraded within cells, thereby eliminating the consequences of mistakes made during protein synthesis. In eukaryotic cells, two major pathways—the ubiquitin-proteasome pathway and lysosomal proteolysis—mediate protein degradation.

The Ubiquitin-Proteasome Pathway

The major pathway of selective protein degradation in eukaryotic cells uses ubiquitin as a marker that targets cytosolic and nuclear proteins for rapid proteolysis (Figure 7.39). Ubiquitin is a 76-amino-acid polypeptide that is highly conserved in all eukaryotes (yeasts, animals, and plants). Proteins are marked for degradation by the attachment of ubiquitin to the amino group of the side chain of a lysine residue. Additional ubiquitins are then added to form a multiubiquitin chain. Such polyubiquinated proteins are recognized and degraded by a large, multisubunit protease complex, called the proteasome. Ubiquitin is released in the process, so it can be reused in another cycle. It is noteworthy that both the attachment of ubiquitin and the degradation of marked proteins require energy in the form of ATP.

Figure 7.39

The ubiquitin-proteasome pathway. Proteins are marked for rapid degradation by the covalent attachment of several molecules of ubiquitin. Ubiquitin is first activated by the enzyme E1. Activated ubiquitin is then transferred to one of several different (more...)

Since the attachment of ubiquitin marks proteins for rapid degradation, the stability of many proteins is determined by whether they become ubiquitinated. Ubiquitination is a multistep process. First, ubiquitin is activated by being attached to the ubiquitin-activating enzyme, E1. The ubiquitin is then transferred to a second enzyme, called ubiquitin-conjugating enzyme (E2). The final transfer of ubiquitin to the target protein is then mediated by a third enzyme, called ubiquitin ligase or E3, which is responsible for the selective recognition of appropriate substrate proteins. In some cases, the ubiquitin is first transferred from E2 to E3 and then to the target protein (see Figure 7.39). In other cases, the ubiquitin may be transferred directly from E2 to the target protein in a complex with E3. Most cells contain a single E1, but have many E2s and multiple families of E3 enzymes. Different members of the E2 and E3 families recognize different substrate proteins, and the specificity of these enzymes is what selectively targets cellular proteins for degradation by the ubiquitin-proteasome pathway.

A number of proteins that control fundamental cellular processes, such as gene expression and cell proliferation, are targets for regulated ubiquitination and proteolysis. An interesting example of such controlled degradation is provided by proteins (known as cyclins) that regulate progression through the division cycle of eukaryotic cells (Figure 7.40). The entry of all eukaryotic cells into mitosis is controlled in part by cyclin B, which is a regulatory subunit of a protein kinase called Cdc2 (see Chapter 14). The association of cyclin B with Cdc2 is required for activation of the Cdc2 kinase, which initiates the events of mitosis (including chromosome condensation and nuclear envelope breakdown) by phosphorylating various cellular proteins. Cdc2 also activates a ubiquitin-mediated proteolysis system that degrades cyclin B toward the end of mitosis. This degradation of cyclin B inactivates Cdc2, allowing the cell to exit mitosis and progress to interphase of the next cell cycle. The ubiquitination of cyclin B is a highly selective reaction, targeted by a 9-amino-acid cyclin B sequence called the destruction box. Mutations of this sequence prevent cyclin B proteolysis and lead to the arrest of dividing cells in mitosis, demonstrating the importance of regulated protein degradation in controlling the fundamental process of cell division.

Figure 7.40

Cyclin degradation during the cell cycle. The progression of eukaryotic cells through the division cycle is controlled in part by the synthesis and degradation of cyclin B, which is a regulatory subunit of the Cdc2 protein kinase. Synthesis of cyclin (more...)

Lysosomal Proteolysis

The other major pathway of protein degradation in eukaryotic cells involves the uptake of proteins by lysosomes. Lysosomes are membrane-enclosed organelles that contain an array of digestive enzymes, including several proteases (see Chapter 9). They have several roles in cell metabolism, including the digestion of extracellular proteins taken up by endocytosis as well as the gradual turnover of cytoplasmic organelles and cytosolic proteins.

The containment of proteases and other digestive enzymes within lysosomes prevents uncontrolled degradation of the contents of the cell. Therefore, in order to be degraded by lysosomal proteolysis, cellular proteins must first be taken up by lysosomes. One pathway for this uptake of cellular proteins, autophagy, involves the formation of vesicles (autophagosomes) in which small areas of cytoplasm or cytoplasmic organelles are enclosed in membranes derived from the endoplasmic reticulum (Figure 7.41). These vesicles then fuse with lysosomes, and the degradative lysosomal enzymes digest their contents. The uptake of proteins into autophagosomes appears to be nonselective, so it results in the eventual slow degradation of long-lived cytoplasmic proteins.

Figure 7.41

The lysosome system. Lysosomes contain various digestive enzymes, including proteases. Lysosomes take up cellular proteins by fusion with autophagosomes, which are formed by the enclosure of areas of cytoplasm or organelles (e.g., a mitochondrion) in (more...)

However, not all protein uptake by lysosomes is nonselective. For example, lysosomes are able to take up and degrade certain cytosolic proteins in a selective manner as a response to cellular starvation. The proteins degraded by lysosomal proteases under these conditions contain amino acid sequences similar to the broad consensus sequence Lys-Phe-Glu-Arg-Gln, which presumably targets them to lysosomes. A member of the Hsp70 family of molecular chaperones is also required for the lysosomal degradation of these proteins, presumably acting to unfold the polypeptide chains during their transport across the lysosomal membrane. The proteins susceptible to degradation by this pathway are thought to be normally long-lived but dispensable proteins. Under starvation conditions, these proteins are sacrificed to provide amino acids and energy, allowing some basic metabolic processes to continue.