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Home , Golgi apparatus, Lysosome, Protein targeting, Translocon

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 , such as mitochondria in eukaryotic cells. and, in the case of photosynthetic organisms, , which generate chemical energy. Other organelles include , which are sites of Objectives degradation for (a kind of eukaryotic trash can), and After this chapter, you should be able to , which house oxidative reactions that break down (catabolize) fatty acids and other small molecules. • explain how the architecture of eukaryotic cells demands mechanisms The of eukaryotic cells also contains an 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 , 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 . and directs them to other organelles, to the cytoplasmic membrane that surrounds the , or to be secreted to the outside of the cell. The ER, the • explain how the 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 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 targeting. Each of the subcellular compartments target membranes. of the cell contains unique proteins. How do these proteins get to their Chapter 16 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 depends on the identity of transport across the compartment. Proteins destined for membranes the nucleus are translated in the cytoplasm and later translocated, in a folded state, nuclear through nuclear pores. Proteins destined pore nucleus for chloroplasts and mitochondria are synthesized on 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, , 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 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 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 tra cked to the into the ER . 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 tra cked to the cytoplasm. mitochondria. No Yes Yes No

The protein is tra cked to the The protein is tra cked 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 , while bound to a protein with a nuclear localization sequence, interacts with the hydrogel, reshaping it so as to allow the 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 , which is a complex that spans the ER membrane and provides a physical channel through which the unfolded, nascent can cross the ER membrane. Translation resumes at its normal rate once the complex is docked with the translocon and the signal recognition particle and signal recognition particle receptor dissociate from the complex. The nascent peptide is then translocated through the channel as it is exported from the ribosome in a process called co-translational translocation. Chapter 16 Protein Targeting 5

Figure 4 Proteins enter the 2 secretory pathway via co- 1 5’ translational translocation, a small ribosomal process mediated by the signal subunit 3’ recognition particle and other large ribosomal protein factors subunit mRNA nascent Proteins that enter the secretory pathway 3’ peptide ER signal bear a signal sequence that directs them sequence to the ER. The synthesis of such proteins 3 5’ initiates in the cytoplasm (State 1). As the signal sequence of the nascent peptide 3’ emerges from the ribosome (State 2), it is recognized by the signal recognition particle (SRP in the figure), which binds to both the signal sequence and the ribosome, signal recognition particle slowing translation (State 3). The signal (SRP) recognition particle carries the entire 4 ribosome-nascent chain complex to the ER 5’ membrane, where the signal recognition particle binds to the signal recognition 5 5’ particle receptor (SRP receptor) in the ER 3’ membrane (State 4). The entire complex is then transferred to the translocon, at which 3’ time the SRP and SRP receptor dissociate, allowing translation to resume (State 5). cytoplasm SRP receptor Translation continues, and the nascent protein is translocated in an unfolded ER membrane state through the translocon and into the ER lumen. The is cleaved ER lumen from the protein by a peptidase (State 6). translocon When translation terminates, the ribosome dissociates from the ER membrane and the nascent protein is released into the ER lumen 5’ (State 7). Proteins with transmembrane domains enter the secretory pathway via 6 the same mechanism, except instead of 7 being released into the ER lumen, their transmembrane domains laterally exit the 3’ translocon, moving sideways into the ER membrane (not shown). cytoplasm

ER membrane

ER lumen

Proteins translated by ER membrane-attached ribosomes can be either soluble (i.e., free-floating) proteins that are imported into the ER lumen (Figure 4) or transmembrane proteins that are inserted into the ER membrane. Soluble proteins simply cross the ER membrane during translation, after which they are released into the ER lumen, where the signal sequence is removed by a peptidase (). Transmembrane proteins are translocated through the translocon until a transmembrane α-helix is reached. Instead Chapter 16 Protein Targeting 6

Figure 5 Targeting sequences (A) COO- direct proteins to particular COO- + cellular compartments NH3 Targeting sequences can either be signal NH + continuous stretches of amino acids, as 3 sequence in the case of the signal sequence (A) or (B) discontinuous patches of amino acids that COO- come together when the protein folds (B). COO- targeting + sequence NH3 + NH3 patch

of passing into the ER lumen, each transmembrane α-helix laterally exits the translocon and enters the ER membrane. Transmembrane α-helices contain stretches of hydrophobic amino acids that are recognized by the translocon and trigger the lateral exit of the helix into the membrane. Some proteins have additional targeting sequences, and in those cases the sequences can function sequentially (Figure 5). For example, one sequence could dictate entry into the ER, while a second, independent sequence can dictate retention in the ER, preventing the protein from being sent on to the cytoplasmic membrane, which might otherwise be its final destination (Figure 6). As an example, imagine a secreted protein that is a dimer (i.e., a protein whose folded structure consists of two polypeptide subunits). Here, an ER retention signal could be used for “quality control” to ensure that only correctly assembled dimers are secreted. In this case the ER retention signal might be covered up by a second monomer once the protein assembles into a correctly folded dimer. With the retention signal buried, the assembled protein in its mature form can then proceed to the cytoplasmic membrane for from the cell. On the other hand, if the protein fails to assemble properly, then the ER retention signal remains exposed, and the protein is not trafficked to the cytoplasmic membrane, instead remaining in the ER. The potassium leak channel, which we discussed in the previous chapter, is an example of a protein that uses this quality-control mechanism. The

COO-

“enter the secretory pathway” COO- - + COO NH3

NH + hidden retention NH + 3 + “remain in the ER” 3 signal NH3 - exposed retention signal COO

Retained in ER Secreted from cell Figure 6 Some proteins contain a retention sequence to ensure proper assembly Shown is a protein with a signal sequence and a retention sequence. The blue signal sequence directs the protein into the lumen of the ER, where the signal sequence is removed by cleavage. A second sequence, shown in red, causes the protein to be retained in the ER, preventing it from progressing through the secretory pathway. When the protein forms a dimer, however, the ER retention signal becomes concealed. When the retention signal is hidden, it is no longer recognized, allowing the protein dimer to progress through the secretory pathway and be secreted from the cell. Chapter 16 Protein Targeting 7 potassium channel is a tetramer (consisting of four polypeptide subunits). The tetrameric structure is assembled in the ER, and an ER retention signal is used to ensure that incorrectly assembled channels are not sent to the cytoplasmic membrane. Each potassium channel monomer contains a particular peptide sequence (Arg-Lys-Arg) that signals its retention in the ER, but when the monomers assemble to form a tetramer, this peptide sequence is covered up by other monomers, preventing it from being recognized and thus allowing the assembled tetramer to exit the ER and be trafficked to the cytoplasmic membrane. On the other hand, if the tetramer fails to assemble correctly, the ER retention signal remains exposed and the channel protein remains in the ER.

The secretory pathway How do proteins migrate from the ER to the Golgi and from the Golgi to other organelles, such as the lysosome and the cytoplasmic membrane, or to be secreted to the outside of the cell? Broadly speaking, the targeting of proteins to each of these organelles or to the is accomplished via a common series of steps known as the secretory pathway, at the heart of which are small membrane vesicles (secretory vesicles) (Figure 7). The vesicles that traffic proteins within the secretory pathway are loaded with cargo proteins from the lumen of one compartment, and they discharge their cargo into the lumen of a second compartment. Secretory vesicles bud off from the membrane of one compartment and fuse with the membrane of another. Vesicles carry cargo from the ER to the Golgi and from the Golgi to various destinations, including , lysosomes, and the cytoplasmic membrane. By default, proteins with only an ER signal sequence will ultimately be trafficked to the cytoplasmic membrane. An additional sequence is generally required for retention in

Figure 7 Proteins in the secretory cytoplasm extracellular space pathway are transported in vesicles nuclear envelope between the ER, Golgi apparatus, and cytoplasmic membrane ER cytoplasmic membrane

secretory vesicle

Golgi apparatus Chapter 16 Protein Targeting 8

cytoplasm 3 cytoplasmic lumen membrane

transmembrane protein donor compartment

2

1

vesicle

cytoplasm extracellular space

Figure 8 topology is conserved during vesicular trafficking The orientation of membrane proteins relative to the cytoplasm is conserved during vesicular transport. This figure shows an example of a membrane protein that contains a region (colored red) that is exposed to the lumen of the donor compartment prior to transport to the cytoplasmic membrane. As the secretory vesicle buds from the donor compartment membrane, the red region remains oriented towards the interior of the vesicle (State 1). In the secretory vesicle this topology is conserved, and the red region of the protein remains on the interior of the vesicle (State 2). When the vesicle fuses with the cytoplasmic membrane, the red region of the protein is exposed to the extracellular space (State 3). At no point is the red region of the protein exposed to the cytoplasm. Similarly, any portion of a membrane protein that is exposed to the cytoplasm is always exposed to the cytoplasm, even during transport. the ER or Golgi or for trafficking to other compartments, such as lysosomes. During the process of budding and fusion, the topology of the protein is maintained (Figure 8). For example, if a transmembrane protein begins the transport process with a domain in the lumen of the donor compartment, that domain will face the lumen of the target compartment. If that transmembrane protein is transported to the cytoplasmic membrane, the domain that previously faced the lumen will face the extracellular space. This also means that any part of a protein that is exposed to the cytoplasm will remain exposed to the cytoplasm for the entire vesicular transport process. The conservation of topology is important for the function of many proteins.

Coat proteins promote budding and select protein cargo The formation of secretory vesicles requires a protein coat that polymerizes around the budding vesicle. The proteins coating a secretory vesicle play critical roles in both physically forming the vesicle and in establishing cargo selection. Vesicles bud off as coated vesicles that have a cage of proteins covering their cytoplasmic surface. Three major types of coated vesicles, named according to their protein coats, are , COPI (“cop one”), and COPII (“cop two”). Each type participates in a particular route of the transport process for a particular set of cargo molecules. For example, COPII vesicles participate in the movement of cargo from the ER to the Chapter 16 Protein Targeting 9 Golgi apparatus, whereas clathrin mediates the import of proteins from the outside to the inside of the cell across the cytoplasmic membrane. Protein coats have two major functions. First, they deform the membrane and introduce curvature to form a vesicle. Second, they concentrate and select for the appropriate cargo proteins. The formation of a vesicle requires the membrane to curve, thereby bringing negatively charged phospholipids into close proximity, an energetically unfavorable state. The introduction of curvature is achieved in part by neutralizing the negative charge on the phospholipid head groups, making it more favorable for the membrane to be deformed. Curvature is also introduced by the shape of the coat proteins themselves; some, like clathrin, are known to have intrinsic curvature and to polymerize into a partial sphere. The tight binding of the coat protein to the membrane and the polymerization of the coat protein causes the membrane to deform. Polymerization of coat proteins is a highly favorable process, and the energy released during polymerization offsets the energetic cost of deforming the membrane. Coat proteins also select cargo. Coats accomplish this by interacting with proteins that are to be enriched in the vesicle. These interactions may be mediated through transmembrane proteins called adaptor proteins, which connect the cargo inside the lumen of the vesicles with a particular protein coat. The adaptor protein helps to specify which cargo molecules are recruited by a particular protein coat. Adaptor proteins in turn recognize and bind to specific sequences in cargo proteins.

SNARE proteins target vesicles to specific destinations and facilitate membrane fusion Vesicles must recognize their correct target membranes; because there are so many membranes in the cell, a vesicle is likely to encounter many Figure 9 SNAREs specify vesicle compartment 1 destinations and facilitate t-SNARE membrane fusion Coat proteins facilitate vesicle formation; coat protein they also select particular cargoes and are associated with specific v-SNAREs that v-SNARE identify the vesicle origin. The v-SNAREs in turn selectively bind to their cognate cargo t-SNAREs on destination membranes. The paired SNAREs facilitate membrane fusion, Budding Docking Fusion and their specific interaction ensures that cargoes are delivered to the correct compartments.

v-SNARE

t-SNARE

compartment 2 Chapter 16 Protein Targeting 10 potential targets. Proteins called SNAREs determine which vesicles fuse with which compartments (Figure 9). Specificity in targeting is ensured because vesicles display v-SNAREs (vesicle SNAREs) on their surface that identify them according to their origin and type of cargo; target membranes display complementary t-SNAREs (target SNAREs) that recognize specific v-SNAREs. The v-SNAREs are packaged together with the coat proteins during the budding of transport vesicles. Contact with a target membrane can trigger coat disassembly. In addition to their role in targeting vesicles to the correct compartments, SNAREs play a role in membrane fusion (Figure 10). Specifically, the highly favorable interactions between cognate v- and t-SNAREs are used to overcome the unfavorable electrostatic repulsion that occurs when two membranes are brought together during fusion. v-SNAREs pair with their cognate t-SNAREs to form a stable bundle of α-helices that forces the two membranes into close apposition; the favorable energetics of the SNARE pairing is used to overcome the unfavorable processes both of expelling water molecules from the interface and of bringing charged phospholipid head groups together. It is hypothesized that the phospholipids of the two interacting leaflets then flow between the membranes to form a connecting stalk. This rearrangement relieves the unfavorable head-to- head interactions of the previous structure. Next, the of the two other

1 v-SNAREs in the vesicle bind to 2 Water is squeezed from between the two 3 Stalk formation complementary t-SNAREs in the membranes target membrane

vesicle

v-SNARE water

target t-SNARE membrane

4 Hemi-fusion 5 Fusion

Figure 10 SNARE proteins allow vesicles to identify and fuse with specific target membranes Chapter 16 Protein Targeting 11 leaflets contact each other, forming a new bilayer that widens the fusion zone; this is known as the hemi-fusion state. Finally, rupture of the new bilayer completes fusion of the vesicle and the target membrane.

Summary Eukaryotic cells have an intricate architecture, with multiple membrane- encased compartments. The presence of multiple membranes necessitates mechanisms for delivering proteins to their proper destinations during or after their release from the ribosome. A key decision point occurs on the ribosome, when nascent proteins that display a signal sequence at their N-terminus are targeted to the endoplasmic reticulum; those that lack a signal sequence are released into the cytoplasm. Proteins that are released into the cytoplasm can remain in the cytoplasm or travel to the nucleus or to particular organelles, such as mitochondria and chloroplasts. For example, proteins with a nuclear localization sequence are recognized by nuclear import receptors, which escort them through channels in the nucleus called nuclear pores. The pores are filled with filamentous proteins that form a gel-like sieve that excludes proteins that are not in a complex with a transport receptor. Proteins that have a signal sequence at their N-terminus are recognized by the signal recognition particle, which binds to the signal sequence as it emerges from the ribosome and slows translation of the mRNA. The entire complex of the arrested ribosome and the signal recognition particle then binds to a receptor on the membrane of the ER, which in turn binds to the translocon, a protein-conducting channel through which the nascent protein is transported as it is translated. Soluble proteins are released directly into the ER lumen, where their signal sequences are removed by a peptidase. Membrane proteins laterally exit the translocon and enter the ER membrane. Transport of proteins among the ER, Golgi, cytoplasmic membrane, and other compartments occurs via the secretory pathway. Proteins enter the secretory pathway via the ER and are trafficked between the organelles of the secretory pathway in secretory vesicles. Soluble proteins are trafficked within the lumens of these vesicles, whereas transmembrane proteins are embedded in the vesicle membrane. The default destination for proteins in the secretory pathway is the cytoplasmic membrane, and additional signaling sequences are typically needed for proteins to be retained in the ER or Golgi or to be trafficked to other compartments. Coat proteins have the dual function of facilitating vesicular budding and selecting the cargo that enters each vesicle. For vesicles to bud, curvature must be introduced into the membrane, which requires an input of energy. Coat proteins interact favorably with one another and polymerize on the outer surface of a budding vesicle. The energy released during this polymerization is used to deform the membrane and promote budding. Coat proteins also select cargo by binding to transport receptors that bind to specific signal sequences in cargo proteins, enriching the vesicle in appropriate cargo proteins. Chapter 16 Protein Targeting 12 SNARE proteins ensure that vesicles fuse with the appropriate target membranes. Each vesicle bears a particular set of SNARE proteins, called v-SNAREs, which identify the origin of the vesicle. Each v-SNARE has a complementary target SNARE (t-SNARE) on the target compartment. Each particular v-SNARE pairs only with its cognate t-SNARE, ensuring that vesicles only fuse to the appropriate target compartments. SNARE protein pairing also facilitates membrane fusion. The v- and t-SNAREs form highly favorable interactions with one another that draw the vesicular and target membranes close together. The energy released as v- and t-SNAREs interact is used to squeeze water molecules from between the two membranes and to encourage the rearrangement and fusion of the membrane bilayers.