40632_CH03_hochstrasser.qxd 3/26/07 11:45 AM Page 1 3 Intracellular protein degradation Mark Hochstrasser Yale University, New Haven, CT CHAPTER OUTLINE 3.1 Introduction 3.8 Integral membrane proteins that function at the cell 3.2 Overview of the ubiquitin-proteasome system surface are degraded within lysosomes 3.3 Ubiquitin attachment to substrates requires multiple 3.9 Under stressful conditions cytoplasmic proteins can be enzymes engulfed into autophagosomes and degraded in the lysosome 3.4 Substrate recognition in the ubiquitin-ligation system 3.10 What’s next? 3.5 Degradation of proteins by the proteasome 3.11 Summary 3.6 Membrane proteins are degraded by several mechanisms 3.12 Supplement 1: Failures of the ubiquitin-proteasome 3.7 Retrotranslocation from the endoplasmic reticulum back system are a cause of human diseases into the cytoplasm allows degradation of short-lived ER proteins 3.13 Supplement 2: Ubiquitin has many functions beyond the proteasome 1 40632_CH03_hochstrasser.qxd 3/23/07 4:52 PM Page 2 3.1 rapidly even without any change in the rate at Introduction which it is synthesized. For example, progres- Key concepts sion of the cell through the cell cycle requires • Different proteins in a cell have vastly different that certain inhibitors of mitosis be inactivated rates of degradation. The cell can regulate those once the chromosomes are aligned and are ready rates. to be separated to opposite poles. At that point • Regulatory proteins such as transcription factors the sensitivity of the inhibitors to proteolysis and cell cycle regulators often have high rates of suddenly increases and they quickly disappear, degradation to allow rapid changes in their con- centration. allowing the cell cycle to advance. • Misfolded or damaged proteins, which are poten- This example illustrates one of the advan- tially toxic, are also targets for efficient elimina- tages of protein degradation as a form of con- tion. trol: its irreversibility. Elimination of a protein • Most intracellular protein degradation is energy- removes any chance of its being reactivated in- dependent, highly specific, and processive. appropriately, which in the case of the cell cy- cle could be lethal to the cell. It is probably for Cells are densely packed with many different this reason that selective protein degradation kinds of proteins, and these proteins are in a is a common component of regulatory mecha- highly dynamic state, moving from place to nisms that determine the relative timing of place within the cell and associating and disso- events. For example, in the cell cycle, mitosis ciating with other proteins and ligands. In view must follow duplication of the chromosomes, of the energy that a cell expends synthesizing so certain factors required for DNA synthesis and organizing its proteins, it may be surpris- are degraded at the end of S phase and are only ing to learn that it is also constantly degrading synthesized again once mitosis has been com- many of them while they are still perfectly func- pleted. tional. Each has its own characteristic rate of In addition to degrading regulatory pro- degradation, or half-life. Most are normally teins, cells also degrade other types of proteins degraded slowly, with half-lives of many hours, while they are still functional. The purpose is days, or longer. Others, in contrast, have half- usually to remove proteins that the cell no longer lives that are much shorter than the doubling needs. This is often the case with proteins that time of the cell, in some cases being as short as help a cell adapt to a particular set of conditions; a few minutes. once the conditions cease to exist the proteins What purpose does proteolysis within a are degraded. Human muscles, for example, are cell serve, and why are the half-lives of some remodeled depending on how they are used proteins so short? One answer to both these and what energy source is available. Remodeling questions is that proteolysis is a very effective involves proteolysis of one set of structural and form of regulation. The rate at which a protein metabolic proteins and its replacement by an- is degraded has a profound effect on how quickly other specialized for a different purpose. its concentration can be changed following a Another function of intracellular proteol- change in its rate of synthesis. A protein that is ysis is to rid the cell of damaged or potentially degraded slowly will persist in the cell even if harmful proteins. Proteins can misfold or de- the cell stops making it. A rapidly degraded pro- nature, they may fail to assemble properly into tein, however, will quickly disappear after its complexes, or they can be altered by some ab- synthesis is stopped. Thus, rapid proteolysis of normal posttranslational modification. Such a protein allows rapid readjustment of its level aberrant proteins are potentially toxic and need within the cell. For this reason, proteins that to be eliminated. The eukaryotic cell has a re- may need to be activated or inactivated quickly markable ability to distinguish normal from ab- often have high rates of degradation, resulting normal proteins and selectively degrade the in short half-lives. This is true of many cellular latter. When this capacity is compromised, dis- regulatory proteins, such as transcription factors, ease often results. enzymes controlling rate-limiting steps in Several important properties characterize biosynthetic pathways, and cell cycle regula- the degradation of most intracellular proteins. tors. Perhaps most obvious is that it must be exquis- Regulation can also be achieved by abruptly itely specific. Only certain proteins should be changing the rate at which a protein is degraded. proteolyzed rapidly, and those often only under If a protein’s rate of degradation is suddenly in- very specific conditions. It is easy to imagine creased its concentration within the cell will fall the devastation that would result if a protease 2 CHAPTER 3 Intracellular protein degradation 40632_CH03_hochstrasser.qxd 3/23/07 4:52 PM Page 3 that lacked such specificity were let loose within the trafficking of proteins into the lysosome. In a living cell. Proteases of the digestive system, this regard, the central question of how only for example, need to be able to cleave proteins particular proteins are degraded in the lyso- with high efficiency but low specificity, and it some is closely linked to the question of how spe- would be catastrophic if such a protease were cific proteins sort between different cellular released in the cytoplasm. A second feature of compartments. intracellular proteolysis is its dependence on the input of energy. Despite the fact that pep- tide bond cleavage is energetically favorable, 3.2 Overview of the ATP hydrolysis is necessary for efficient degra- dation of most proteins within the cell. This en- ubiquitin-proteasome ergy requirement derives largely from the need system for very high substrate specificity and can be understood in light of what we now know about Key concepts the molecular mechanisms of most cellular pro- • Cellular proteins to be degraded are first cova- teolytic pathways. These mechanisms will be lently linked to a polymer of ubiquitin polypep- the main topic of this chapter. tides; this polyubiquitin chain allows the substrate A final property that distinguishes most in- to bind to the proteasome, where the substrate protein is processively degraded to small peptides. tracellular proteolysis is that it is processive: • The ubiquitin-proteasome system performs most once a protein is selected for proteolysis, it is intracellular proteolysis. completely degraded to small peptides without larger intermediate forms being released. Processive degradation prevents the release Proteolysis of specific proteins in a cell is usu- of large protein fragments that might interfere ally performed by the ubiquitin-proteasome with cell function. For example, many tran- system. The enzymes comprising this system scription factors have their DNA-binding and can, when necessary, select and degrade almost transcriptional activation activities in separate any protein in the cell. Such proteolysis serves protein domains. If the DNA-binding domain a number of purposes. It orchestrates various cel- were proteolytically separated from the activat- lular events—such as critical cell cycle transi- ing domain, it would still be able to bind DNA tions—by eliminating regulatory proteins; it and might compete with the normal factor for removes proteins that the cell no longer needs; regulatory sites in the genome. and it provides an important quality control This chapter will focus on the major mech- function by continuously scanning the cell’s anisms of protein degradation in the eukary- collection of proteins and ridding it of those that otic cell. The majority of regulated degradation are damaged or abnormal. In all cases these en- occurs through the ubiquitin-proteasome sys- zymes act with great specificity, not only iden- tem, which functions in both the cytoplasm and tifying individual proteins from among the the nucleus. In order for a protein to be de- thousands in a cell, but often distinguishing dif- graded by this mechanism, it is first covalently ferent forms of the same protein depending on modified by the attachment of multiple copies factors such as subcellular localization, post- of a small polypeptide called ubiquitin. The translational modification, or inclusion in par- polyubiquitin-modified protein is recognized ticular multiprotein complexes. by an abundant, multisubunit protease called the The system is organized around ubiquitin, proteasome, which degrades the substrate into a small protein that is used as a label to desig- short peptides. The next section introduces the nate proteins for destruction. Ubiquitin is added ubiquitin-proteasome system in greater detail. to proteins in a tightly linked series of reactions Some proteolysis of cellular proteins also (FIGURE 3.1). A protein to be degraded is first rec- occurs in the lysosome, a membrane-enclosed ognized by a specific ubiquitin-protein lig- organelle.