Protein Targeting

16 Protein Targeting To understand the molecular The cells of eukaryotic organisms are architecturally intricate (Figure 1). Goal mechanisms that are They have a nucleus in which the genetic material is stored and transcribed responsible for the sorting of into RNA. They also have specialized organelles, such as mitochondria proteins in eukaryotic cells. and, in the case of photosynthetic organisms, chloroplasts, which generate chemical energy. Other organelles include lysosomes, which are sites of Objectives degradation for macromolecules (a kind of eukaryotic trash can), and After this chapter, you should be able to peroxisomes, which house oxidative reactions that break down (catabolize) fatty acids and other small molecules. • explain how the architecture of eukaryotic cells demands mechanisms The cytoplasm of eukaryotic cells also contains an endoplasmic reticulum to target specific proteins to appropriate (ER), an extensive maze of interconnected spaces (lumena) surrounded by destinations. a membrane that serves as the site for the synthesis of proteins destined for • describe how the decision to import into the ER. The ER is, in turn, connected to the Golgi apparatus, enter either the cytoplasm or the a stack of flattened disks of membrane that receives proteins from the ER endoplasmic reticulum is made on the ribosome. and directs them to other organelles, to the cytoplasmic membrane that surrounds the cell, or to be secreted to the outside of the cell. The ER, the • explain how the nuclear pore can block Golgi, and all of the other individual organelles are surrounded by a single the nuclear entry of some proteins but not others. membrane bilayer, with the exception of the nucleus, mitochondria, and chloroplasts. The nucleus is surrounded by a double membrane bilayer • explain how proteins enter the endoplasmic reticulum. called the nuclear envelope that is contiguous with the endoplasmic reticulum. Mitochondria and chloroplasts are also surrounded by inner • explain the role of vesicles and coat and outer membrane bilayers. proteins in the secretory pathway. • describe how v-SNAREs and t-SNAREs Thus, the ultrastructure of the eukaryotic cell is complex, posing challenging enable vesicles to identify and fuse with questions about protein targeting. Each of the subcellular compartments target membranes. of the cell contains unique proteins. How do these proteins get to their Chapter 16 Protein Targeting 2 Figure 1 Proteins are targeted to different cellular compartments by a variety of mechanisms The mechanism by which a protein is trafficked to a particular cellular compartment depends on the identity of transport across chloroplast the compartment. Proteins destined for membranes the nucleus are translated in the cytoplasm and later translocated, in a folded state, nuclear mitochondrion through nuclear pores. Proteins destined pore nucleus for chloroplasts and mitochondria are synthesized on ribosomes in the cytoplasm transport through and later translocated into the target proteins nuclear pores compartment in an unfolded state. Proteins that are to be secreted and proteins that are destined for the ER, Golgi, lysosome, or cytoplasmic membrane are all translocated ER ribosomes into the ER as they are synthesized. From vesicle the ER, these proteins travel in membrane- bound vesicles to various cellular compartments. transport by Golgi vesicles secreted protein proper destination, be it the cytoplasmic membrane, the lysosome, the mitochondrion, or the chloroplast? How do proteins become embedded in membranes or cross membranes to reach a compartment on the other side or for export out of the cell? Here we examine the ways in which the cell addresses these protein sorting and transport challenges. The first decision is whether to enter the endoplasmic reticulum The journey of a protein to its proper destination is governed by a series of decisions (Figure 2). The initial decision is whether to enter the endoplasmic reticulum. ER entry is determined by the presence or absence of a short stretch of amino acids at the N-terminus of a nascent polypeptide chain as it emerges from the ribosome. These amino acids are known as thesignal sequence, and we will return to them presently. If a signal sequence is absent, the newly synthesized protein is released into the cytoplasm. Such proteins might remain in the cytoplasm (for example, many of the myriad enzymes of the cell). However, particular amino-acid sequences (think of them as addresses) target certain proteins to the nucleus, the mitochondria, or other organelles. Targeting the nucleus Passage into (and out of) the nucleus occurs through specialized channels known as nuclear pores (Figure 3). These protein-lined structures span both the inner and outer membranes of the nuclear envelope. Nuclear Chapter 16 Protein Targeting 3 The mRNA leaves the nucleus, and its translation begins in the cytoplasm. Is there an ER signal sequence? No Yes SRP binds to the nascent ER The translated protein is released signal sequence, halting into the cytoplasm. translation until the ribosome docks to the ER membrane. Is there a nuclear localization signal? Is there a transmembrane domain? No Yes No Yes The translated protein is The translated protein is released embedded into the ER Is there a mitochondrial The protein is tracked to the into the ER lumen. membrane via its targeting sequence? nucleus. transmembrane domain(s). No Yes Is there a second targeting Is there a second targeting sequence? sequence? The protein remains in the The protein is tracked to the cytoplasm. mitochondria. No Yes Yes No The protein is tracked to the The protein is tracked to the cytoplasmic membrane and cytoplasmic membrane. secreted. The protein is retained in the The protein is retained in the lumen of the targeted membrane of the targeted compartment (e.g., ER, Golgi, compartment (e.g., ER, Golgi, lysosome, etc.). lysosome, etc.). Figure 2 Proteins are targeted to particular cellular locations depending on their targeting sequences Shown is a decision tree that describes how particular sequence(s) affect protein-targeting decisions. pores are large enough (100 nm) that ions and small molecules can freely diffuse through them, but proteins cannot move through the pore without assistance. This assistance takes the form of specialized proteins that act as nuclear import receptors. Figure 3 Proteins enter the cytoplasmic lament nucleus through nuclear pores central pore cytoplasmic ring hydrogel outer nuclear membrane nuclear envelope inner nuclear membrane nuclear basket nuclear ring Chapter 16 Protein Targeting 4 The nuclear pore is lined with long, flexible, filamentous proteins that interact with each other via backbone-backbone hydrogen bonding and hydrophobic interactions to form a gel-like material known as a hydrogel. Importantly, the hydrogel acts as a sieve, preventing large molecules from passing through the channel. The import receptors recognize and bind to proteins that bear a particular amino-acid sequence known as a nuclear localization sequence. The nuclear import receptor, while bound to a protein with a nuclear localization sequence, interacts with the hydrogel, reshaping it so as to allow the protein complex to traverse the channel of the nuclear pore and enter the nucleus. Signal sequences target proteins to the ER Now let’s consider the case of proteins that do have a signal sequence at their N-termini (Figure 4). The signal sequence is generally a short stretch of hydrophobic amino acids that targets proteins to the ER. The ER serves as the entry point of proteins destined for other organelles. Proteins that are targeted to the lysosome, the Golgi, and the cytoplasmic membrane all enter the ER first. Once inside the ER, proteins do not re-enter the cytoplasm. How does this targeting take place? Proteins that lack a signal sequence are synthesized on ribosomes that are free and untethered in the cytoplasm. However, proteins that contain a signal sequence are synthesized on ribosomes that are associated with the outer surface of the ER. Indeed, the ER can be covered with many such dot-like ribosomes, which impart a rough appearance to its surface in electron micrographs (hence, it is referred to as rough ER). The way this happens is as follows. Since the signal sequence is at the N-terminus, when the mRNA for a protein that is destined for the ER starts to be translated, the signal sequence is the first part of the protein to emerge from the ribosome. The signal sequence in the nascent protein is recognized by the signal recognition particle, which, as we will see, attaches the ribosome to the ER membrane, resulting in a membrane-attached ribosome. The ribosomal subunits, both large and small, are recycled after each round of translation, and depending on which mRNA they happen to translate, they will either become free or membrane- attached. Thus, the two categories of ribosomes are functionally equivalent and draw from a common pool of ribosomal subunits in the cytoplasm. The signal recognition particle does two things (Figure 4). First, binding of the signal recognition particle to the signal sequence of the nascent protein temporarily slows translation. Second, the resulting complex, consisting of the mRNA, ribosome, nascent protein chain, and signal recognition particle, docks to a receptor on the surface of the ER known as the signal recognition particle receptor. Once associated with the ER membrane, the complex is directed to the protein translocation channel or translocon,

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