Lecture 16 Study Guide
1. Describe different types of endoplasmic reticulum and their functions
Rough ER is responsible for the synthesis and assembly of secreted proteins.
Smooth ER aid the formation of steroid hormones.
2. What are microsomes, how are they made, and what are their properties/functional characteristics?
Microsomes are small, closed, vesicles that form when the ER breaks into fragments. They are still capable of protein translocation, protein glycosylation, Ca2+ uptake and release, and lipid synthesis.
3. What is co-translational translocation and post-translational translocation? Which process allows protein import into the ER?
The co-translational transfer process allows protein import into the ER, and creates two spatially separate populations of ribosomes. Membrane-bound ribosomes, attached to the cytosolic side of the ER membrane, are engaged in the synthesis of proteins that are being concurrently translocated across the ER membrane. Free ribosomes, unattached to any membrane, synthesize all other proteins encoded by the nuclear genome. Membrane-bound and free ribosomes are structurally and functionally identical. They differ only in the proteins they are making at any given time.
Some proteins are completely synthesized in the cytosol as precursors before they are imported into the ER, demonstrating that translocation does not always require ongoing translation. This is termed post-translational translocation. Post-translational protein translocation is more common across the yeast ER membrane and the evolutionarily related bacterial plasma membrane. In both cases, the Sec61 translocator (called SecY in bacteria) is used as the translocator; its narrow channel means that precursors can only be translocated as unfolded polypeptides. Thus, precursor proteins do not fold after their initial synthesis in the cytosol. Instead, they interact with other cytosolic proteins that prevent precursor folding or aggregation before they engage the Sec61 translocator. These interacting proteins typically are general chaperone proteins, such as those of the hsp70 family, and must dissociate as the unfolded polypeptide is threaded through the translocator.
4. Describe the structure, function, and interactions of the signal recognition particle
The ER signal sequence is guided to the ER membrane by at least two components: a signal-recognition particle (SRP), which binds to the signal sequence, and an SRP receptor in the ER membrane. SRP is a large complex; in animal cells, it consists of six different polypeptide chains bound to a single RNA molecule. While SRP and SRP receptor have fewer subunits in bacteria, homologs of both components are present in all living organisms.
ER signal sequences vary greatly in amino acid sequence, but each has eight or more nonpolar amino acids at its center.
In eukaryotic cells, SRP is a hinged rodlike structure that can wrap along the large ribosomal subunit. The end of SRP that contains the signal sequence-binding pocket is positioned near the ribosomal tunnel through which newly made polypeptides emerge.
When a signal sequence binds, SRP exposes a binding site for an SRP receptor, which is a transmembrane protein complex in the rough ER membrane. The binding of SRP to its receptor brings the SRP-ribosome complex to an unoccupied protein translocator in the ER membrane. The part of SRP bound near the ribosomal tunnel moves to a different site, allowing the translocator to occupy this position. SRP and SRP receptor are then released, and protein synthesis resumes at full speed. The translocator, which is now tightly bound to the translating ribosome, transfers the growing polypeptide chain across the membrane.
How ER signal sequences and SRP direct ribosomes to the ER membrane: The SRP and its receptor act in concert. The SRP binds to both the exposed ER signal sequence and the ribosome, thereby causing translation to slow. The SRP receptor in the ER membrane, which in animal cells is composed of two different polypeptide chains, binds the SRP-ribosome complex and directs it to the translocator. The SRP (in complex with SRP receptor) then moves away from its binding site on the ribosome, which is then occupied by the translocator in the ER membrane. SRP then releases the signal sequence, which inserts into the translocator to initiate polypeptide chain transfer across the lipid bilayer. The SRP and SRP receptor dissociate from each other and are recycled for the next round of protein targeting. Although not shown in the figure, one of the SRP proteins and both chains of the SRP receptor contain GTP-binding domains. Conformational changes that occur during cycles of GTP binding and hydrolysis (discussed in Chapter 15) ensure that SRP preferentially binds a signal sequence in the cytosol and releases it only after SRP successfully engages the SRP receptor at the ER membrane. The energy of GTP hydrolysis is therefore used to impart directionality to the cycle of SRP-mediated protein targeting.
13). How can SRP bind specifically to so many different sequences?
The answer has come from structures of one of the SRP proteins, which shows that the signal sequence-binding site is a large hydrophobic pocket enriched in methionines (Figure 12-19B).
Because methionines have unbranched, flexible side chains, the pocket is sufficiently plastic to accommodate different hydrophobic signal sequences of various sizes and shapes.
5. What is the structure of the ER translocator? What is its core component? How do different structural components of the translocon contribute to its function?
Aqueous pore
– Gated by a short α-helix
• Why is this important?
– Pore can also open along a seam in its side
• Allows translated peptide access to the hydrophobic membrane
The translocator was shown to form a water-filled channel across the membrane through which the polypeptide chain passes. The core of the translocator, called the Sec61 complex, is built from three subunits that are highly conserved from bacteria to eukaryotic cells. The structure of the Sec61 translocator revealed that 10 alpha helices surround a central channel. The channel is plugged by a short a helix that keeps the translocator closed when it is idle. It is important to keep the channel closed to prevent ions, such as Ca2+ from leaking out of the ER. During translocation, the plug moves out of the way so the polypeptide can pass through the channel.
The Sec61 translocator only opens for proteins containing a signal sequence. The ability of the Sec61 translocator to recognize signal sequences provides a proofreading step to ensure that only proteins truly intended for the ER are allowed to enter. Cryo-electron microscopy structures of the Sec61 translocator before and after signal sequence recognition show that the signal sequence wedges into a lateral gate, or seam, in Sec61 with its N-terminus facing the cytosol. Insertion of the signal sequence at this lateral gate widens the central channel and releases the plug. The open translocator then readily accommodates the segment of polypeptide following the signal sequence inside the channel. The signal sequence, which is hydrophobic, laterally exits the gate into the membrane where it is cleaved off by signal peptidase and then rapidly degraded to amino acids by other proteases in the ER membrane and cytosol. As this mechanism illustrates, the lateral gate in the Sec61 translocator provides the access route from Sec6l's central channel into the hydrophobic core of the membrane. In addition to its role in recognition of signal sequences, the lateral gate guides the integration of transmembrane proteins into the ER. Once the signal sequence has opened the Sec6l translocator and threaded the ensuing polypeptide into the channel, translocation occurs concurrently with continued translation. During translocation, the polypeptide tunnel inside the ribosomal large subunit is aligned with the channel within the Sec61 translocator (Figure 12-23B). This configuration provides a continuous path for the polypeptide from the peptidyl-transferase center in the ribosome, where new amino acids are added to the growing protein chain, to the ER lumen 15 nm away. In this way, the energy used for polypeptide elongation is indirectly harnessed to also drive translocation across the ER membrane.
When translation terminates, the C-terminus of the polypeptide is released from the ribosome and slips through the Sec6l translocator, whose plug returns to close the channel. Thus, the entire process of ER import, from signal sequence recognition by SRP to translocation through the Sec61 translocator, occurs co-translationally before the polypeptide has a chance to fold. This pathway provides one solution to the problem of how to move a large protein across a membrane barrier without leakage of much smaller ions and metabolites during the process.
Translocator+accessory proteins=translocon
Chaperone proteins are necessary to keep the polypeptide chain in an unfolded state prior to and during translocation. Similarly, a source of energy is required to provide directionality to the transport and to pull the protein into the cytosol. Finally, a translocator is necessary.
6. What happens to proteins lacking a signal sequence?
Cannot be imported into the ER. Stay in the cytosol for the rest of translation. If they lack other "address labels," they'll stay in the cytosol permanently. However, if they have the right labels, they can be sent to the mitochondria, chloroplasts, peroxisomes, or nucleus after translation.
7. What enzyme removes signal sequences? Where does this happen?
Signal peptidase removes signal sequences in the ER
8. Describe 3 methods through which a single-pass transmembrane protein can be produced
Stop sequence causes discharge of both cleaved start sequence and protein into membrane.
In the simplest case, a transmembrane protein contains a single transmembrane segment that will ultimately be embedded in the lipid bilayer as a membrane-spanning alpha helix. When this transmembrane segment emerges from the ribosome during synthesis, SRP recognizes its hydrophobic alpha-helical features as a signal sequence and brings this ribosome to the Sec6l translocator at the ER membrane. The transmembrane segment then inserts into the lateral gate of the Sec61 translocator, which is the same site where signal sequences bind. The orientation in which the transmembrane segment inserts into the lateral gate determines whether the protein segment preceding or the one following the transmembrane segment is moved across the membrane into the ER lumen. If the N-terminus is short and unfolded, orientation of the transmembrane segment depends on features of the polypeptide chain such as the distribution of nearby charged amino acids and the length of the transmembrane segment. If the preceding N-terminal segment is long and stably folded, it does not cross the membrane through the narrow Sec61 channel. In this case, the C-terminal segment that is still being synthesized, and therefore unfolded, is translocated across the membrane.
Many transmembrane proteins contain large N-terminal lumenal domains. In this case, an N-terminal signal sequence is used to initiate translocation, just as for a soluble protein. In this way, the N-terminus of the mature polypeptide is committed to the ER lumen by the signal sequence, and the remainder of the polypeptide begins translocation through the Sec61 translocator. When a hydrophobic segment in the polypeptide emerges from the ribosome, it inserts into the lateral gate to gain access to the lipid bilayer. Because the hydrophobic segment is more stable in the membrane than in the aqueous channel, it exits the channel laterally, translocation stops, and the rest of the protein is synthesized on the cytosolic side of the ER membrane.
Type I: N-terminus on the extracellular side of the membrane; removed signal peptide
Type II: N-terminus on the cytoplasmic side of the membrane; transmembrane helix located close to the N-terminus, where it works as an anchor
Type III: N-terminus on the extracellular side of the membrane; no signal peptide.
Type I proteins are anchored to the lipid membrane with a stop-transfer anchor sequence and have their N-terminal domains targeted to the ER lumen during synthesis.
Type II and III are anchored with a signal-anchor sequence, with type II being targeted to the ER lumen with its C-terminal domain, while type III have their N-terminal domains targeted to the ER lumen.
9. Know how different combinations of start-transfer signal sequences, stop-transfer signal sequences, and the location of positive and negative amino acids near these sequences affect the final structure of a transmembrane protein
Another way that proteins are attached to the membrane is by glycosylphosphatidylinositol (GPI) anchor that is covalently linked to the C-terminus of some proteins destined for the plasma membrane. GPI-anchored proteins are initially made with an N-terminal signal sequence to direct them to the ER and a hydrophobic segment very close to the C-terminus. This hydrophobic segment is selectively recognized by a transamidase enzyme in the ER membrane that simultaneously cleaves off the hydrophobic segment and attaches a preformed GPI anchor to the rest of the protein..
The attachment of a GPI anchor to a protein in the ER. GPI-anchored proteins are targeted to the ER membrane by an N-terminal signal sequence (not shown), integrated into the membrane, and processed by signal peptidase similarly to a single-pass transmembrane protein (see Figure 12-
27). Immediately after the completion of protein synthesis, the precursor protein remains anchored in the ER membrane by a hydrophobic C-terminal sequence of 15-20 amino acids; the rest of the protein is in the ER lumen.
Within less than a minute, a transamidase enzyme in the ER cleaves the protein from its membrane-bound C-terminus and simultaneously attaches the new C-terminus to an amino group on a preassembled GPI intermediate.
The sugar chain contains an inositol attached to the lipid from which the GPI anchor derives its name. It is followed by a glucosamine and three mannoses.
The terminal mannose links to a phosphoethanolamine that provides the amino group to attach the protein through an amide bond. The signal that specifies this modification is contained within the hydrophobic C-terminal sequence and a few amino acids adjacent to it on the lumenal side of the ER membrane; if this signal is added to other proteins, they too become modified in this way. Because of the covalently linked lipid anchor, the protein remains membrane-bound, with all of its amino acids exposed initially on the lumenal side of the ER and eventually on the exterior of the plasma membrane.
The ER resident protein protein disulfide isomerase (PDI) catalyzes the oxidation of free sulfhydryl (SH) groups on cysteines to form disulfide (S-S) bonds (Figure 12-31). Almost all cysteines in protein domains exposed to either the extracellular space or the lumen of organelles in the secretory and endocytic pathways are disulfide bonded. Disulfide bonds stabilize the folded state of a protein, enabling it to better withstand a harsh, variable, and chaperone-free extracellular environment. Because proteins often contain multiple cysteines, they sometimes pair incorrectly. PDI resolves this problem by rearranging the disulfide bonds in a protein until it is correctly folded. This is possible because PDI enzymes are capable of operating in reverse to reduce incorrectly paired disulfides of immature proteins. The ER lumen contains multiple members of the PDI family, some of which are specialized for reducing disulfide bonds to fully unfold misfolded proteins that need to be translocated back to the cytosol for degradation (discussed later). All PDI enzymes are therefore oxidoreductases that can catalyze either the formation or breakage of disulfide bonds in their client proteins. The formation of disulfide bonds relies on maintaining an oxidizing environment in the ER lumen. Disulfide bonds form only very rarely in domains exposed to the cytosol because of the reducing environment there.
Proteins enter the ER lumen as unfolded polypeptides. They must therefore fold and assemble into their correct three-dimensional structures just as newly made proteins in the cytosol must fold (discussed in Chapter 3). To meet this demand, the lumen of the ER contains a high concentration of resident chaperones and other protein-folding catalysts. These ER resident proteins contain an ER retention signal of four amino acids at their C-terminus that is responsible for retaining the protein in the ER (see Figure 12-13; discussed in Chapter 13, p. 768).
The protein BiP, a member of the hsp70 family of chaperone proteins, is a major component of the ER folding machinery. We have already discussed how BiP pulls proteins post-translationally into the ER through the Sec61 ER translocator. Like other chaperones (discussed in Chapter 6), BiP recognizes incorrectly folded proteins, as well as protein subunits that have not yet assembled into their final oligomeric complexes. It does so by binding to exposed hydrophobic amino acid sequences that would normally be buried in the interior of correctly folded or assembled polypeptide chains. The bound BiP both prevents the protein from aggregating and helps keep it in the ER (and thus out of the Golgi apparatus and later parts of the secretory pathway). BiP hydrolyzes ATP to shuttle between high- and low-affinity polypeptide-binding states. In this way, BiP periodically lets go of its substrate proteins to allow them an opportunity to fold, and then re-binds them if folding is not yet achieved.
N-linked protein glycosylation in the rough ER. (A) Almost as soon as a polypeptide chain enters the ER lumen, it is glycosylated on target asparagine amino acids. The precursor oligosaccharide (shown in color) is attached only to asparagine side chains in the sequences Asn-X-Ser and Asn-X-Thr (where X is any amino acid except proline). These sequences occur much less frequently in glycoproteins than in nonglycosylated cytosolic proteins. Evidently there has been selective pressure against these sequences during protein evolution, presumably because glycosylation at inappropriate sites would interfere with protein folding. The five sugars in the gray box form the core region of this oligosaccharide. For many glycoproteins, only the core sugars survive the extensive oligosaccharide trimming that takes place in the Golgi apparatus (Movie 13.4). (B) The precursor oligosaccharide is transferred from a dolichol lipid anchor to the asparagine as an intact unit in a reaction catalyzed by a transmembrane oligosaccharyl transferase enzyme complex. One copy of this enzyme is associated with each protein translocator in the ER membrane. Oligosaccharyl transferase contains 13 transmembrane a helices and a large ER lumenal domain that contains binding sites for the nascent protein and dolichol-oligosaccharide.
The asparagine binds a tunnel that penetrates the enzyme interior. There, the amino group of the asparagine is twisted out of the plane that stabilizes the otherwise poorly reactive amide bond, activating it for reaction with the dolichol-oligosaccharide.
The N-linked oligosaccharides are by far the most common oligosaccharides, being found on 90% of all glycoproteins. Less frequently, oligosaccharides are linked to the hydroxyl group on the side chain of a serine, threonine, hydroxylysine, or hydroxyproline amino acid. The first sugar of these O-linked oligosaccharides is added in the ER. N-linked and O-linked oligosaccharides undergo extensive processing, modification, and extension in the Golgi apparatus (Chapter 13), producing the diversity of oligosaccharide structures observed on mature glycoproteins.
