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Module 9 Worksheet
Terms in this set (43)
Protein insertion into the mammalian ER membrane is typically
Signal sequences that direct proteins to the ER membrane are:
Stretches of hydrophobic amino acids located generally located near the amino terminus of the protein
___________ provides the driving force for translocating a polypeptide chain into the ER post-translationally.
ATP hydrolysis by BiP
N-linked oligosaccharides are:
A) Added in the cis Golgi and modified in the trans Golgi
B) Added in the trans Golgi and modified in secretory vesicles
C) Added in the ER and modified in the Golgi
D) Added in the Golgi and modified in the ER
Added in the ER and modified in the Golgi
GPI anchored membrane proteins are membrane associated by
a covalently attached lipid
The topology of membrane proteins can often be predicted by computer programs that identify ________________________________________ topogenic segments.
During N-glycosylation of proteins, an oligosaccharide precursor is first synthesized with _______sugar residue (s) and this preformed precursor is later transferred to the nascent polypeptide chain.
Which of the following is a lectin?
D) prolyl isomerase.
Proteins that do not fold properly in the ER lumen are degraded in the cytosol by
Sorting of protein to mitochondria is
Tom/Tim protein complexes are involved in
protein translocation into mitochondria
Sequences that target proteins to mitochondria are located at
the N-terminus of the precursor protein
Protein import into the mitochondrial matrix is supported by energy input from
A) ATP hydrolysis by chaperone proteins in the cytosol.
B) ATP hydrolysis by chaperone proteins in the mitochondrial matrix.
C) the proton-motive force across the inner mitochondrial membrane.
D) all of the above
all of the above
Many peroxisomal matrix proteins are imported as
The nuclear pore complex allows for
A) passive diffusion of smaller molecules.
B) import of proteins.
C) active transport of very large molecules.
D) all of the above
all of the above
The nuclear transport receptor can bind to:
A) FG nucleoporins.
C) basic nuclear localization signals in cargo proteins.
D) all of the above
all of the above
The signal recognition particle (SRP) binds to the ________________ soon after it appears outside the ribosome.
The ______________________ binds to the signal peptide soon after it appears outside the ribosome.
signal recognition particle (SRP)
The amino acid sequences that target proteins to chloroplasts, mitochondria and nuclei have the following property in common:
When added in the proper context, they are sufficient to direct the targeting of a foreign proteins into the respective organelles
Glycosylation of proteins inside the endoplasmic reticulum does not involve:
a His residue on the protein
ER Type-I transmembrane proteins possess all of the following but NOT:
A) cleavable signal sequence.
B) internal signal-anchor sequence.
C) internal stop-transfer sequence.
D) N terminus-out of ER lumen and C terminus-inside the lumen topology.
N terminus-out of ER lumen and C terminus-inside the lumen topology
Elucidate three different pathways for targeting proteins to the mitochondrial inner membrane.
Path A involves the importing of cytochrome oxidase subunit CoxVa. In Path A, CoxVa precursor which is marked with an N-terminus matrix-targeting sequence is imported by the outer membrane receptor Tom40 after being detected by the outer membrane receptor Tom20/22. After this, it then passes through an inner membrane receptor Tim23/17. Throughout this process, the matrix-targeting sequence is removed and a hydrophobic sequence on the CoxVa makes it so the protein is laterally transferred into the inner membrane bilayer.
Path B involves the importing of ATP synthase subunit 9. In Path B, ATP synthase subunit 9 is similarly transported like in path A (using Tom40 and Tim23/17), but the integration of this protein into the inner membrane does differ. ATP synthase subunit 9 must interact with an inner-membrane protein called Oxa1.
Path C which involves the importing of ADP/ATP antiporter. In path C, proteins (such as ADP/ATP antiporter) are recognized by the Tom70/Tom22 receptor in the outer membrane. The proteins then pass through Tom40 receptor in the outer membrane and then through Tim22/Tim54 with the help of intermembrane proteins Tim9/10. This receptor complex (Tim22/18/54) allows for the incorporation of the imported ADP/ATP antiporter.
What is the meaning of "quality control in the ER?" Describe the unfolded-protein response. What is the fate of unassembled or misfolded proteins present in the ER?
"Quality control in the ER" means that the protein needs to be modified or folded before it can leave the ER and go to the Golgi apparatus. The steps are as follows: 1) the unfolded protein response is activated when there is a high accumulation of unfolded proteins 2) the response senses that the ER is in a stressed state and so it activates an intracellular signaling pathway 3) this intracellular signaling pathway communicates to the cytosol and nucleus that the ER is stressed 4) the intracellular signaling pathway ultimately slows down the rate of translation, ups the creation of proteins that take part in the unfolded protein response, and increases degradation of accumulated, unfolded proteins.
Thus, the fate of unassembled or misfolded proteins present in the ER is that they are tagged via ubiquitination and then degraded by the proteasome.
To what extent do peroxisomal matrix protein import and peroxisomal membrane protein import share the same machinery?
Peroxisomal matrix protein import and peroxisomal membrane protein import do not share the same machinery. We know this because there have been studies on cells affected by Zellweger syndrome which is the defection of peroxisomal matrix protein import. If matrix protein import and membrane protein import did share the same machinery, we would expect that the peroxisomal membrane proteins would be indirectly affected by this syndrome. Yet because it is not and because there is a standard composition of peroxisomal membrane proteins, this indicates that they do not share the same import machinery.
Explain how unidirectional nature of protein export and import through nuclear pores is facilitated by the G protein Ran?
The G protein Ran interacts with a nuclear transport receptor called importin to facilitate the export and import of proteins. When importin interacts with Ran in the nucleoplasm, a conformational change occurs that knocks off the protein to be imported. Then the Ran and attached importin return back to the cytoplasmic side and the attached GTP is hydrolyzed to GDP, decreasing the affinity of Ran for importin, and consequently knocking importin off. The lone Ran and GDP can then go back into the nucleoplasm, find another GTP and continue the cycle of binding to importin all over again.
How are proteins imported into the thylakoids of chloroplasts?
Four separate pathways for transporting proteins from the stroma into the thylakoid have been identified. All four pathways have been found to be closely related to analogous transport mechanisms in bacteria.
1.) Transport of plastocyanin and related proteins into the thylakoid lumen from the stroma occurs by an SRP-dependent pathway that uses a translocon similar to SecY, the bacterial version of the Sec61 complex.
2.) A second pathway for transporting proteins into the thylakoid lumen involves a protein related to bacterial protein SecA, which uses the energy from ATP hydrolysis to drive protein translocation through the SecY translocon.
3.) A third pathway, which targets proteins to the thylakoid membrane, depends on a protein related to the mitochondrial Oxa1 protein and the homologous bacterial protein.
4.) Thylakoid proteins that bind metal-containing cofactors follow another pathway into the thylakoid lumen. The unfolded precursors of these proteins are first targeted to the stroma, where the N-terminal stromal-import sequence is cleaved off, and the protein then folds and binds its cofactor. A set of thylakoid-membrane proteins assists in translocating the folded protein and bound cofactor into the thylakoid lumen. This process is powered by the H+ electrochemical gradient normally maintained across the thylakoid membrane.
Describe the basic function of 3 different cytosolic proteins required for translation into the ER, mitochondria, and peroxisomes, respectively.
For translation to the ER, there is a cytosolic protein called SRP (signal recognition particle) that recognizes and then directs certain proteins to the ER. For translation to the mitochondria, there is a cytosolic protein called Hsp70 which aids in the partial folding and directing of proteins into the mitochondrial matrix. For translation to the peroxisomes, there is a cytosolic protein receptor called Pex5 that directs PTS1-bound proteins into the peroxisomes.
1. The following results were obtained in early studies on the translation of secretory proteins. Based on what we now know of this process, explain the reason why each result was observed.
A) An in vitro translation system consisting only of mRNA and ribosomes resulted in secretory proteins that were larger than the identical protein when translated in a cell.
B) A similar system that also included microsomes produced secretory proteins that were identical in size to those found in a cell.
C) When the microsomes were added after in vitro translation, the synthesized proteins were again larger than those made in a cell.
a. In the absence of ER membranes, the entire protein is translated and the ER signal sequence remains on the protein.
b. When translation occurs in the presence of ER-containing microsomes, the protein is translated into the lumen of the microsomes. Following this process, the signal sequence is cleaved producing a smaller protein.
c. Translation and translocation across the ER membrane are simultaneous processes. If they do not occur at the same, the protein is not properly imported into the ER where the signal sequence can be cleaved (although there are some examples of post-translational translocation).
Describe the source or sources of energy needed for unidirectional translocation across the membrane in (a) cotranslational translocation into the endoplasmic reticulum (ER); (b) post-translational translocation into the ER; (c) translocation into the mitochondrial matrix.
a. The energy source for cotranslational translocation comes from the translation process itself—in other words, the nascent chain is pushed through the translocon channel. Please note, however, that as translation is completed a portion of the newly synthesized protein still resides within the translocon. This portion is drawn into the ER lumen rather than being pushed.
b. In post-translational translocation, the newly synthesized polypeptide chain is drawn through the translocon by an energy input from ATP hydrolysis by BiP. BiP is luminal protein of the ER and is a member of the Hsc70 family of molecular chaperones. BiP-ATP activates by binding to the Sec63 complex that in turn binds to the Sec61 translocon complex. Activated BiP is enzymatically active and cleaves ATP to ADP plus Pi. It is BiP-ADP that binds to the entering, unfolded nascent chain. Sequential binding of BiP-ADP to the nascent chain serves to block any sliding of the chain back and forth in the translocon and to ratchet the nascent chain
through the translocon.
c. Translocation into the mitochondrial matrix occurs through a bipartite Tom/Tim complex in which Tom is the outer membrane translocon and Tim is the inner membrane translocon. Three energy inputs are required. First, ATP hydrolysis by a cytosolic Hsc70 chaperone keeps the newly synthesized mitochondrial precursor protein unfolded in the cytosol. Second, ATP hydrolysis by multiple ATP-driven matrix Hsc70 chaperones may serve to pull the translocating protein into the matrix. Matrix Hsc70s interact with Tim44 and hence may be analogous to the BiP/Sec63 interaction at the ER membrane. Third, energy input from the H+ electrochemical gradient or proton-motive force is required. The inside-negative membrane electric potential may serve to electrophorese the amphipathic matrix-targeting sequence toward the matrix.
Translocation into most organelles usually requires the activity of one or more cytosolic proteins. Describe the basic functions of three different cytosolic factors required for translocation into the ER, mitochondria, and peroxisomes, respectively.
SRP (signal recognition particle) acts as a cycling cytosolic factor for the translocation of ER targeted proteins. It binds to both the signal sequence and SRP receptor, a heterodimer associated with the ER membrane. In doing this, SRP initiates ribosome binding to ER membranes and positions the nascent chain proximal to the translocon. Both SRP and the SRP receptor are GTPases. The unfolded nascent chain then translocates. Cytosolic Hsc70 functions as a cytosolic factor required for protein translocation into mitochondria. It acts as a molecular chaperone to keep the post-translationally targeted mitochondrial precursor protein in an open, extended conformation. At least two different cytosolic proteins are required for translocation of peroxisomal matrix proteins. These are Pex5, the soluble receptor protein for matrix proteins containing a C-terminal PTS1 targeting sequence, and Pex7, the soluble receptor protein for matrix proteins containing an N-terminal PTS2 targeting sequence. A different receptor, Pex19, is required for peroxisomal membrane proteins.
Describe the typical principles used to identify topogenic sequences within proteins and how these principles can be used to develop computer algorithms. How does the identification of topogenic sequences lead to prediction of the membrane arrangement of a multipass protein? What is the importance of the arrangement of positive charges relative to the membrane orientation of a signal-anchor sequence?
Many membrane proteins are embedded in the membrane by virtue of transmembrane alpha-helical segment(s). Such segments can be referred to as topogenic sequences. These segments share general principles or properties. They tend to be about 20 amino acids long, a length sufficient to span the membrane, and hydrophobic, an appropriate property for a sequence embedded in the hydrophobic lipid bilayer. Application of these principles through computer algorithms is predictive. In brief, amino acid sequences of polypeptides may be scanned for hydrophobic segments of about 20 amino acids long. Each amino acid may be assigned a hydrophobic index value based on relative solubility in hydropbobic media versus water, and these values then can be summed by a computer for all 20 amino acid segments of a protein. Segments exceeding a threshold value are expected to be topogenic transmembrane segments. Internal signal anchor and stop-transfer anchor segments similarly can be identified. Such sequences alternate within a multipass membrane protein. Because of this, the overall arrangement of the protein can be predicted as described in detail in the text.
An abundance of misfolded proteins in the ER can result in the activation of the unfolded-protein response (UPR) and ER-associated degradation (ERAD) pathways. UPR decreases the abundance of unfolded proteins by altering gene expression of what type of genes? What is one manner in which ERAD may identify misfolded proteins? Why is dislocation of these misfolded proteins to the cytoplasm necessary?
The UPR pathway up-regulates transcription of protein chaperones. It is thought that the timing of glycosylation modifications is one manner in which misfolded ER proteins are identified. Dislocation into the cytoplasm is necessary because the proteolytic machinery for these ER proteins is located in the cytoplasm.
Temperature-sensitive yeast mutants have been isolated that block each of the enzymatic steps in the synthesis of the dolichol-linked oligosaccharide precursor for N-linked glycosylation. Propose an explanation for why mutations that block synthesis of the intermediate with the structure dolichol-PP-(GlcNAc)2Man5 completely prevent addition of N-linked oligosaccharide chains to secretory proteins, whereas mutations that block conversion of this intermediate into the completed precursor—dolichol-PP-(GlcNAc)2Man9Glc3—allow the addition of N-linked oligosaccharide chains to secretory glycoproteins.
The seven-sugar intermediate is synthesized by sugar addition to cytosolic-facing dolichol phosphate. The intermediate is flipped from the cytosolic face of the ER membrane to the luminal face. Further sugar additions then occur within the lumen of the ER. Short forms of the intermediate are on the wrong side of the membrane to add to nascent polypeptides within the ER lumen. Incomplete adductants within the ER lumen are located appropriately to N-glycosylate nascent polypeptide.
Name four different proteins that facilitate the modification or folding of secretory proteins within the lumen of the ER. Indicate which of these proteins covalently modifies substrate proteins and which brings about only conformational changes in substrate proteins.
Several proteins facilitate the modfication or folding of secretory proteins within the ER. These include signal peptidase, BiP, oligosaccharyl transferase, various glycosidases, calnexin and calreticulun, protein disulfide isomerase, peptidyl-prolyl isomerase, and others. Of these, BiP and peptidyl-prolyl isomerase act to facilitate conformation changes. Protein disulfide isomerase facilitates the making/breaking of disulfide bonds to ensure correct protein folding. Calreticulin and calnexin are lectins that bind to glycoproteins during folding. The others all directly support the covalent modification of proteins within the ER lumen.
Describe what would happen to the precursor of a mitochondrial matrix protein in the following types of mitochondrial mutants: (a) a mutation in the Tom22 signal receptor; (b) a mutation in the Tom70 signal receptor; (c) a mutation in the matrix Hsp70; and (d) a mutation in the matrix signal peptidase.
a. Tom22 together with Tom20 act as outer mitochondrial membrane receptor proteins for N-terminal matrix targeting sequences. A defective Tom22 receptor protein would result in accumulation of mitochondrial matrix targeted proteins in the cytosol, possibly followed by their turnover within the cytosol.
b. Tom70 signal receptor is an outer mitochondrial membrane protein recognizing multipass mitochondrial membrane proteins that have internal signal sequences. Mutation in Tom70 will have no immediate effect on mitochondrial matrix protein import, as Tom70 does not recognize this class of protein.
c. Matrix Hsc70 has a role in the folding of matrix proteins. Also, it is one source of energy for powering translocation. Defective matrix Hsc70 should result in clogging the Tom/Tim translocon complex with incompletely translocated proteins.
d. Retention of the matrix targeting N-terminal signal sequence because of a defective matrix signal peptidase might well result in defective folding of the imported protein. The sequence normally is removed.
Describe the similarities and differences between the mechanism of import into the mitochondrial matrix and the chloroplast stroma.
On the whole, protein import into the mitochondrial matrix and the chloroplast stroma, topologically equivalent locations, is by functionally equivalent mechanisms. Functionally analogous proteins mediate each process. However, the proteins are not homologous, indicating a separate evolutionary origin of mitochondria and chloroplasts. Energetically, unlike the situation for mitochondria, there is no need for a membrane electrochemical gradient for import into chloroplasts. Presumably, stromal Hsc70 pulls proteins into the stroma.
Design a set of experiments using chimeric proteins, composed of a mitochondrial precursor protein fused to dihydrofolate reductase (DHFR), that could be used to determine how much of the precursor protein must protrude into the mitochondrial matrix in order for the matrix-targeting sequence to be cleaved by the matrix-processing protease.
This is basically a molecular ruler question. How many amino acids must span the Tom/Tim complex to expose the matrix-targeting sequence to the matrix-processing protease? DHFR in the presence of the drug methotrexate is locked into a folded state. A chimeric mitochondrial protein with folded DHFR fails to translocate fully into the mitochondria matrix. Instead, it is stuck in the Tom/Tim complex. The number of amino acids between the matrix targeting sequence and the folded DHFR sequence could be varied to provide a molecular ruler. Any unfolded N-terminal DHFR sequence must be included within the ruler. With respect to channel length, an overestimate will result from this approach, as the matrix targeting sequence must be spaced out from Tom/Tim to be accessible for cleavage.
Peroxisomes contain enzymes that use molecular oxygen to oxidize various substrates, but in the process, hydrogen peroxide—a compound that can damage DNA and proteins—is formed. What is the name of the enzyme responsible for the breakdown of hydrogen peroxide to water? What is the mechanism of the import of this protein into the peroxisome, and what other proteins are involved?
Catalase is responsible for breaking down H2O2 to H2O. Catalase, like most other peroxisome-localized enzymes, contains a peroxisome-targeting sequence (PTS1) consisting of three amino acids, serine-lysine-leucine, at its C-terminus. This PTS1 is recognized and binds in the cytosol to the Pex5 receptor. The catalase-Pex5 heterodimer moves to the peroxisome membrane, where it interacts with the Pex14 receptor located on the membrane. In this position, the complex interacts with three membrane proteins—Pex2, Pex10, and Pex12—that facilitate the translocation of catalase into the peroxisome.
Suppose that you have identified a new mutant cell line that lacks functional peroxisomes. Describe how you could determine experimentally whether the mutant is primarily defective for insertion/assembly of peroxisomal membrane proteins or matrix proteins.
Separate mechanisms are used to import peroxisomal matrix and membrane proteins. Hence, mutations can selectively affect one or the other. Either can result in the loss of functional peroxisomes. One approach to determining whether the mutant is primarily defective in insertion/assembly of peroxisomal membrane proteins or matrix proteins is to use antibodies to ask by microscopy if either class of proteins localize to "peroxisomal" structures (e.g., peroxisome ghosts). An alternate approach is cell fractionation, in which the assay determines whether the appropriate proteins are present in a membrane organelle fraction.
The nuclear import of proteins larger than 40 kDa requires the presence of what amino acid sequence? Describe the mechanism of nuclear import. How are nuclear transport receptors able to get through the nuclear pore complex?
The NLS. The nuclear import receptor binds to the NLS on the cargo molecule and brings it into the nucleus. Here, the receptor binds to Ran-GTP, causing release of the cargo. The receptor is thought to interact with the FG repeats that are commonly found on nuclear pore complex proteins, moving from one to the next as it passes through the nuclear pore.
Why is localization of Ran-GAP in the nucleus and Ran-GEF in the cytoplasm necessary for unidirectional transport of cargo proteins containing an NES?
Ran-guanine nucleotide-exchange factor (Ran-GEF) must be present in the nucleus and Ran-GAP must be in the cytoplasm for unidirectional transport of cargo proteins across the nuclear pore complex. When the Ran is bound to GTP, it has high affinity for cargo proteins. During nuclear export, Ran-GTP picks up cargo proteins in the nucleus and carries them to the cytoplasm. To be able to release the cargo on the cytoplasmic side, GTP must be hydrolyzed to GDP, and this process is stimulated by Ran-GAP. Once translocated back into the nucleus, Ran needs to be in the GTP-bound state to pick up more cargo. Ran-GEF in the nucleus stimulates the exchange of GDP for GTP, and this process of export can start again.
Which of the following is true about targeting of a secretory proteins?
Binding of SRP to the signal peptide and the ribosome temporarily accelerates protein synthesis.
The newly synthesized polypeptides include a signal peptide at their carboxyl termini.
The signal peptide is cleaved off inside the mitochondria by signal peptidase.
The signal recognition particle (SRP) binds to the signal peptide soon after it appears outside the ribosome.
The signal sequence is added to the polypeptide in a posttranslational modification reaction.
The signal recognition particle (SRP) binds to the signal peptide soon after it appears outside the ribosome.
List the post-translational modifications that occur in the ER. Why are bacteria often a poor choice for the production of proteins for therapeutic purposes?
Glycosylation: covalent addition and processing of carbohydrates. N-linked or O-linked oligosaccharides are carbohydrates that are added to proteins to form glycoproteins, which are commonly used for cell-surface signaling.
Formation of disulfide bonds: Disulfide bonds help stabilize the tertiary and quaternary structure of proteins, and these bonds are formed in the ER by the oxidation reaction of sulfhydryl (thiol) groups between two cysteine residues.
Proper folding of polypeptide chains and assembly of multi-subunit proteins: Sequential actions of ER lumen proteins, protein folding enzymes, enables efficient folding of polypeptide chains into mature, folded proteins.
Specific proteolytic cleavage: Unassembled or misfolded proteins are transported to the cytosol for degradation. ER membrane proteins recognizes the incorrectly folded protein and target it for transport into the cytosol via the process of dislocation.
Bacteria are often a poor choice for production of therapeutic proteins because prokaryotic cells do not have an ER. Although the bacteria may be able to produce the unfolded polypeptide chain as precursor for a therapeutic protein, the bacteria would have no way to efficiently and quickly make sure the protein was correctly folded or bound to carbohydrates or to other protein subunits. It is unlikely that very much of the mature folded protein could be produced efficiently by the bacteria. Eukaryotic cells with an ER are much more effective at ensuring correct and efficient protein folding and other post-translational modifications.
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