BC-465 Exam 2 Study Questions

R-smad interacts with?

Co-smad

What is a false statement about the smooth ER?

Co-translational protein translocation

Describe composition characteristics of a MAP pathway and give one example.

A cascade of three kinases that relay signals into a cell. There Raf-MEK-ERK pathway is a well characterized example.

Compare structures and functions between chaperones and chaperonins regard to their roles in facilitating protein folding.

-In molecular biology, molecular chaperones are proteins that assist the covalent folding or unfolding and the assembly or disassembly of other macromolecular structures. Chaperones are present when the macromolecules perform their normal biological functions and have correctly completed the processes of folding and/or assembly. The chaperones are concerned primarily with protein folding. One major function of chaperones is to prevent both newly synthesised polypeptide chains and assembled subunits from aggregating into nonfunctional structures. It is for this reason that many chaperones, but by no means all, are heat shock proteins because the tendency to aggregate increases as proteins are denatured by stress. In this case, chaperones do not convey any additional steric information required for proteins to fold. However, some highly specific 'steric chaperones' do convey unique structural (steric) information onto proteins, which cannot be folded spontaneously. Such proteins violate Anfinsen's dogma,[4] requiring protein dynamics to fold correctly. -Chaperonins are barrel shaped proteins that provide favourable conditions for the correct folding of other proteins, thus preventing aggregation. They prevent the misfolding of proteins, which prevents diseases such as Mad Cow Disease. Newly made proteins usually must fold from a linear chain of amino acids into a three-dimensional form. Chaperonins belong to a large class of molecules that assist protein folding, called molecular chaperones.[1] The energy to fold proteins is supplied by adenosine triphosphate (ATP). Chaperonin proteins may also tag misfolded proteins to be degraded.

The signal recognition particle (SRP) most commonly:

-Recognizes signal sequence which are composed of a several hydrophobic amino acids -Binds the signal sequence as it emerges from the ribosome -introduces a pause in translation -Binds to the SRP-receptor on the ER membrane

As a result of T-cell activation, NF-AT migrates from the cytoplasm to the nucleus to regulate gene expression. Describe the basic molecular reactions that lead to this outcome. The beast immunosuppressive drugs (cyclosporine and FK506) used in human organ transplantation block lymphocyte proliferation by inhibiting calcineurin. What is the cellular mechanism for this application?

-The T-cell antigen receptor complex binding to a peptide antigen bound to the MHC complex on the surface of the antigen presenting cell activates lck, a non receptor membrane tyrosine kinase (belongs to the src kinase family) -The active lck phosphorylates ICAMS tyrosine on the several cytosolic chains of the TCR complex, generating binding sites for proteins carrying the SH2 domain. -ZAP-70, a protein kinase carrying two SH2 domains, is then recruited to the TCR complex and is activated by the active lck. -The LAT (Linker for activation of T-cell) is a transmembrane protein and is then phosphorylated by the activated ZAP-70, thus generating a binding site for PLCy leading to activation of PLCy. -The activated PLCy acts on the PIP2 molecules at the inner leaflet of the PM to produce DAG and IP3, two lipid-derived second messengers. -The release of IP3 acts on the ER associated IP3 receptor, a IP3-gated Ca2+ channel, to cause Ca2+ release, which then activates calcineurin, a protein phosphatase regulated by CaM. -The NF-AT (nuclear factor of the activated T-cell, a potent transcription factor) is normally phosphorylated and present in the cytoplasm at the resting state. Upon calcineurin activation, NF-AT is dephosphorylated exposing its NLS to be delivered into the nucleus to stimulate T-cell proliferation. There are only 10,000 calcineurin in lymphocytes while other cells have 300,000 (i.e. 30 fold difference). When used at low concentrations, these inhibitors (Cyclosporin and FK506) selectively block NF-AT import by suppressing calcineurin activity, making human heart and liver transplantation feasible.

List five covalent post-translational modifications that can happen to a protein.

1. Acetylation 2.Phosphorylation 3. Prenylation 4.Glycosylation 5. Ampylation

What are the three main classes of proteins that must be separated before they leave the TGN?

1. Those destined for lysosomes (via late endosomes) 2. Those destined for secretory vesicles 3. Those destined for immediate delivery to the cell surface.

How many kinds of ubiquitin modifications can happen to a protein?

3 kinds of ubiquitin modifications can happen to a protein. 1.Mono-Ubiquitination 2.Multi-Ubiquitination 3.Poly-Ubiquitination

If you smell a rose for a while, it becomes less aromatic. What is the cellular mechanism for this phenomenon?

A ligand (odorant molecule) binding to its receptor not only activates the positive stimulatory signals but also initiates several feedback pathways to desensitize the smell. For example, alleviated levels of cAMP activate PKA, which in turn phosphorylates the ligand bound receptor. Additionally, the Beta/Gamma complex dissociated from the trimeric GTPase, can activate olfactory receptor kinase (ORK) the also phosphorylates the light bound receptor. Arrestin binding to the phosphorylated receptor prevent further activation of trimeric GTP even in the presence of the fragrance.

Insulin Receptor Substrate interacts with?

Activated insulin receptor

Cadherin interacts with?

Beta-catenin

The COPII complex that packages cargo into transport vesicles is assembled when the Sar-1 protein:

Binds GTP

Calcium interacts with?

Calmodulin

How does calmodulin contribute to regulate target protein functions in response to a transient increase in intracellular calcium concentration?

Calmodulin binds to calcium ions and undergoes conformational changes. The calcium bound calmodulin then binds to the target protein and regulates the target function.

What are the cellular factors that help to stabilize the nascent hydrophobic peptide emerging from ribosomes?

Chaperones and chaperonins are proteins that assist the covalent folding or unfolding and the assembly or disassembly of other macromolecular structures. Chaperones are present when the macromolecules perform their normal biological functions and have correctly completed the processes of folding and/or assembly. The chaperones are concerned primarily with protein folding. One major function of chaperones is to prevent both newly synthesised polypeptide chains and assembled subunits from aggregating into nonfunctional structures.

Vesicle budding is associated with coat proteins. Wat is the role of coat proteins in vesicle budding? How are coat proteins recruited to membranes? What kind of molecules are likely to be included or excluded from newly formed vesicles? What is the best-known example of a protein involved in vesicle pinching off?

Coat proteins play two roles in vesicle budding: 1) they provide a scaffold that establishes membrane curvature; 2) they interact with cargo or cargo receptors to provide enrichment of certain molecules in the bud. Small GTPases recruit coat proteins to membrane. A well-characterized example is the recruitment of COPII coat protein (sec13/31) by Sar1 GTPase (named sec24 in yeast). Arf GTPases recruit clathrin and COP I. AP complexes are also capable of recruiting clathrin proteins. Vesicle formation is a highly selective process. First of all, vesicles are enriched in specific cargo molecules that are recognized by specific membrane receptors. Also, newly formed vesicles are programmed for subsequent fusion events by the selective inclusion of v-SNAREs and GTPases(Rabs) in their membrane. Dyanmin is the well-known protein (a GTPase) to have a role in pinching off vesicles at the cell surface and at the TGN.

Which of the following is not characteristic of the olfactory system?

Each olfactory neuron expresses a few kinds of seven helix odorant receptors.

List Three possible outcomes if a newly synthesized protein mis-folded

Facilitated chaperones or chaperonins at cost of ATP, a misfolded protein can fold again into the correct conformations. A misfolded protein can also be degraded by proteasome after polyubitination. Some misfolded proteins form insoluble aggregates that lead to malfunction of cells-causes of prion diseases

All the molecules that enter early endosomes ultimately reach late endosomes, where they become mixed with newly synthesized acid hydrolases and end up in lysosomes. True or false. Why.

False. Many molecules that enter early endosomes are diverted from the journey to late endosomes and lysosomes; they are recycled instead from early endosomes back to the plasma membrane via transport vesicles.

Any particle that is bound to the surface of a phagocyte will be ingested by phagocytosis. True or false. Why.

False. Not all particles that bind are ingested. Phagocytes have a variety of specialized surface receptors that are functionally linked to the phagocytic machinery of the cell. Only those particles that bind to these specialized receptors can be phagocytosed.

Once a secretory vesicle is properly positioned beneath the plasma membrane, it will immediately fuse with the membrane and release its contents to the cell exterior. True or false and why.

False. Once positioned underneath the plasma membrane, a secretory vesicle waits until the cell receives an appropriate signal - often a rise in Ca2+ concentration - before fusing with the membrane and releasing its contents.

Free calcium ions can travel a long distance in cells since they are very small in size. True of false, why.

False. There are many cellular proteins, such as calmodulin, that have high binding affinity to calcium, and hence function to sequester free calcium ions from the cytoplasm.

Transferrin Receptor interacts with?

Fe-bound transferrin

Describe the major steps and cellular elements involved for a protein carrying a NES to enter the cytoplasm from the nucleus.

Fror cargo to exit the nucleus the it first has to get in the export signal in the nucleus. This is done by using Ran-GEF which exchanges GDP for GTP which allows for the export receptor to enter the NPC. Once in the cargo with the NES is loaded onto the export receptor and Ran-GAP hydrolyzes GTP to GDP allowing the Ran-GDP to dissociate from the receptor.

SOS interacts with?

Ras-GDP

Nucleus transport is a gated process. What does it mean regarding the sizes of proteins across the NPC?

If the protein is smaller than 40 kD than it can freely diffuse but if the protein is larger than this then gated transport is needed.

The ER is an important site of "quality control" for newly synthesized proteins. What is meant by "quality control" in this context? What proteins and their functions are typically involved in the processing of newly synthesized proteins with the ER?

Improperly folded (or oligomerized) proteins fail to exit the ER. In other words, ER exit is dependent on protein (folding) quality. Proteins unable to exit the ER are retrotranslocated back to the cytosol, where they are degraded in the cytosol in an ubiquitin-dependet, proteasome- dependent manner. A transferase enzyme necessary for N-glycosylation of appropriate proteins is involved to mark newly synthesized proteins. Protein-folding chaperones, such as BiP, help to stabilize unfolded proteins. A glucosyl transferase recognizes improperly folded proteins and adds a glucose moiety to the oligosaccharide chain to label them. These improperly folded proteins retained within the ER through binding to Calnexin until they are folded correctly.

Patients with Oguchi's disease have been found to have defects in the gene for rhodopsin-specific kinase. After a flash of bright light these individuals recover their night vision (become dark adapted) very slowly (night blindness). Explain the molecular basis of the visual response in these patients' rod cells in comparison to that in normal individuals.

In patients with oguchi's disease, deficiency in rhodopsin specific kinases significantly delays the recovery process. The activated rhodopsin continues to stimulate cGMP phosphodiesterase activity and thus the rod cells remain hyperpolarized (the active form). Consequently, they are unable to sense (see) additional photons- night blindness for a long time

Iron (Fe) is an essential trace metal that is needed by all cells. Iron is taken into cells via a two-component system. The soluble protein transferrin circulates in the bloodstream, the transferrin receptor is a membrane protein that is continually endocytosed and recycled back to the plasma membrane. Fe ions bind to transferrin receptor at neutral pH but not at acidic pH. Transferrin binds to the receptor at neutral pH only when it has bound an Fe ion, but it binds to the receptor at acidic pH even in the absence of bound iron. From there properties, describe how iron is taken up, and discuss the advantage of the elaborate scheme.

In the absence of bound Fe, transferrin does not interact with its receptor and continues to circulate in the bloodstream until it catches an Fe ion. Once iron is bound, the iron-transferrin complex can bind to the transferrin receptor on the surface of a cell and be endocytosed. Under the acidic conditions of the endosome, the transferrin releases its iron but remains associated BC465/565 spring 2017 with the transferrin receptor, which is recycled back to the cell surface. The neutral pH of the extracellular environment causes the receptor to release the transferrin into the circulation, where it can pick up another Fe ion to repeat the cycle. This system allows cells to take up iron efficiently; transferrin is only endocytosed when it has an Fe bound.

The transferrin and transferrin receptor are required for iron uptake that is needed by all cells. This is achieved by receptor-mediated endocytosis. Describe what happens to a) an Fe ion; b) a transferrin; c) a transferrin receptor in this process.

In the absence of bound Fe, transferrin does not interact with its receptor and continues to circulate in the bloodstream until it catches an Fe ion. Once iron is bound, the iron-transferrin complex can bind to the transferrin receptor on the surface of a cell and be endocytosed. Under the acidic conditions of the endosome, the transferrin releases its iron but remains associated BC465/565 spring 2017 with the transferrin receptor, which is recycled back to the cell surface. The neutral pH of the extracellular environment causes the receptor to release the transferrin into the circulation, where it can pick up another Fe ion to repeat the cycle. This system allows cells to take up iron efficiently; transferrin is only endocytosed when it has an Fe bound.

What is the molecular mechanism that cause the I-cell disease?

Inclusion-cell (I-cell) disease, also referred to as mucolipidosis II (ML II), is part of the lysosomal storage disease family and results from a defective phosphotransferase (an enzyme of the Golgi apparatus). This enzyme transfers phosphate to mannose residues on specific proteins, and serves as a marker for them to be targeted to lysosomes within the cell. Without this marker, the proteins are instead excreted outside the cell—the default pathway for proteins moving through the Golgi apparatus. Lysosomes cannot function without these proteins, which function as catabolic enzymes for the normal breakdown of substances (e.g. oligosaccharides, lipids, and glycosaminoglycans) in various tissues throughout the body (i.e. fibroblasts). As a result, a buildup of these substances occurs within lysosomes because they cannot be degraded, resulting in the characteristic I-cells, or "inclusion cells." These cells can be identified under the microscope. In addition, the defective lysosomal enzymes normally found only within lysosomes are instead found in high concentrations in the blood.

Calmodulin?

Is a ubiquitous protein in eukaryotic cells, and also binds multiple Ca++ in a cooperative fashion

Protein glycosylation in the ER serves all of the following functions except

It reversibly activates or inactivates metabolic enzymes

List a retrieval sequence for soluble proteins resident to the ER lumen. How does the presence of a retrieval sequence on a soluble ER protein result in its retrieval from the cis-Golgi complex?

KDEL at the C-terminus is a sequence feature of soluble ER luminal proteins. Retreival of a normally ER luminal proteins from the CGN is a COP1-dependent process. The KDEL receptor is an ER membrane protein bridging interactions between KDEL proteins and COP1 coat proteins at CGN for the formation of retrieval vesicles.

The Endoplasmic Reticulum has many soluble resident proteins in the lumen. These proteins are sometimes co-packaged into COPII coated vesicles and delivered to the Golgi apparatus. Describe the cellular mechanism that brings these proteins back to the ER lumen.

KDEL at the C-terminus is a sequence feature of soluble ER luminal proteins. Retreival of a normally ER luminal proteins from the CGN is a COPI-dependent process. The KDEL proteins and COPI coat proteins at CGN for the formation of retrieval of vesicles.

Multivesicular bodies form on the pathway to:

Late endoscopes

Mannose-6-phosphate receptor interacts with?

Lysosomal hydrolase

Describe molecular connection between aberrant protein folding and the development of certain neurodegenerative diseases such as Alzheimers's disease.

Many misfolded proteins involved in disease contain one or more mutations that destabilize the correct fold and/or stabilize a misfolded state. In Alzheimers the formation of Beta-sheets cause aggregation to form amyloid fibrils. These are very stable structures and accumulate in the tissues and cause problems.

Describe the major components and basic steps for unidirectional translocation across the membrane in (a) co-translational translocation into the ER; (b) post- translational translocation into the ER.

Most proteins that are secretory, membrane-bound, or reside in the endoplasmic reticulum (ER), golgi or endosomes use the co-translational translocation pathway. This process begins with the N-terminal signal peptide of the protein being recognized by a signal recognition particle (SRP) while the protein is still being synthesized on the ribosome. The synthesis pauses while the ribosome-protein complex is transferred to an SRP receptor on the ER in eukaryotes, and the plasma membrane in prokaryotes. There, the nascent protein is inserted into the translocon, a membrane-bound protein conducting channel composed of the Sec61 translocation complex in eukaryotes. After SRP binds the SRP receptor, GTP get hydrolyzed to GDP to allow SRP to dissociate. Even though most secretory proteins are co-translationally translocated, some are translated in the cytosol and later transported to the ER/plasma membrane by a post-translational system. In prokaryotes this requires certain cofactors such as SecA and SecB. This pathway is poorly understood in eukaryotes, but is facilitated by Sec62 and Sec63, two membrane-bound proteins. In addition, proteins targeted to other destinations, such as mitochondria, chloroplasts, or peroxisomes, use specialized post-translational pathways. Also, proteins targeted for the nucleus are translocated post-translation. They pass through the nuclear envelope via nuclear pores.

In which of the following types of cells would you expect to find a very extensive rough endoplasmic reticular network?

Plasma (antibody secreting) cells

The inside of the nucleus is characterized by high levels of?

Ran-GEF

Describe distribution characteristics of Ran-GTP and Ran-GDP molecules in the nucleus and cytoplasm. What is the biological significance of these distribution characteristics? What is the molecular mechanism that contributes to maintaining these features?

Ran-GTP (active) is normally found in the nucleus and Ran-GDP (inactive) in the cytoplasm. These distinct distributions of Ran-GTP and Ran-GDP are important to determine directionality for protein transport into and out of the nucleus. This is achieved by localization of Ran-GAP mainly in the cytoplasm and Ran-GEF solely in the nucleus. When a Ran-GTP moves out of the nucleus, the cytosolic Ran-GAPs stimulate its hydrolysis and quickly convert it into Ran-GDP. Once a Ran-GDP enters the nucleus, the nucleus-associated Ran-GEFs facilitate exchange of GDP for GTP, converting Ran-GDP into Ran-GTP.

Exocytosis is a process by which cells

Release of substances from the cell via vesicles

When you switch from a bright room to a dark room, it takes a few seconds for your eyes to be able to see things around you. What is the molecular and cellular mechanism of this phenomenon?

Rod cells are responsible for sensing dim light and are mostly in the excited form (hyperpolarized) when in a bright room. They need to return to the ground state (recovery) in order to be able to sense dim light again. This is done by Rhodopsin kinases that phosphorylate only the active form of Rhodopsin (as a result of photon absorption). Then, arrestin binding to the phosphorylated rhodopsin prevent the activated rhodopsin from stimulating the pathway that breaks down cGMP. Consequently, the continuously active guanylyl cyclases make up more cGMP to open cGMP gated channels and then cells become depolarized returning back to the resting state and ready to sense photons. This process takes a few seconds to occur in normal eyes.

Describe the role of rhodopsin kinases in visual adaptation/recovery.

Rod or cone cells are responsible for sensing light. At the resting state in the dark, these cells have continuously active guanalyl cyclases that make cGMP out of GTP to operate (open) cGMP-gated channels. As a result, these cells are depolarized, which causes secretion glutamate, a neurotransmitter, as a signal. Photon absorption by rhodopsin activates the cellular pathway that leads to decreases in cGMP concentration and closure of cGMP-gated channels. Subsequently, the cells become hyperpolarized, which inhibits glutamate secretion. These changes in glutamate secretion signal to the neuron cells that are in contact with the cells. Rhodopsin Kinases are associated with the disk membrane by a C-terminal farnesyl group. To recover from the activated state (hyper polarized membrane), rhodopsin kinases phosphorylate only the active form of rhodopsin (after photon absorption). Then, arrestin binding to the phosphorylated rhodopsin prevent the activated rhodopsin from stimulating the pathway that breaks down cGMP. As a result, the continuously active guanilyl cylcases make up mor cGMP to open cGMP-gated channels and then cells become depolarized returning back to the resting state ready to sense photons.

Frequently, vesicles traveling from the ER to the Golgi or between Golgi membranes, fuse to form larger vesicles that then travel together as one. This "homotypic fusion" is the result of the interaction of ________ proteins.

SNARE

How can different MAP kinase pathways carry out specific responses when many of the upstream components can cross-activate each other?

Scaffolding of the MAPKKK, MAPKK, and MAPK can organize the signaling pathways into a linear signaling pathway. Different cells express different proteins that are targeted by the kinases.

What are the biological functions of bride of sevenless (Boss) and son of sevenless (Sos)? Describe their roles in the signaling pathway they are involved in.

Sevenless is a tyrosine kinase receptor expressed on the surface of ommatidium cell R7 which interacts with the bride of sevenless, the ligand expressed on R8. Ligand (Boss) binding to receptor (sevenless) activates the cytoplasmic kinase domain, leading to recruitment of a GEF (son of Sevenless) to the plasma membrane where it activates Das (a small GTPase) that is membrane-anchored via prenylation.

ER import signal sequence interacts with?

Signal Recognition particle

A macrophage ingests the equivalent of 100% its plasma membrane each half hour by endocytosis. What is the rate at which membrane is returned by exocytosis?

Since the surface area and volume of a macrophage do not change significantly over this time, the rate of exocytosis must also equal to 100% of the plasma membrane each half hour.

What are the effectors that are directly activated by receptor serine/threonine kinases?

Smalls

What is the importance of the arrangement of charges relative to the membrane orientation of a signal-anchor sequence?

The ER membrane is characteristic of more negatively charged at the cytosolic side; more positively charged at the luminal side. A signal-anchor sequence with an opposing charge distribution at its ends would prefer incorporation of the transmembrane sequence in such a way that the positively charged end preferentially associates with the cytosolic side and the negatively charged end with the luminal leaflet.

Calcium ion is a unique second message that plays an important role in cell signaling. What are the distribution and concentrations characteristics of calcium ion inside and outside a mammalian cell? What are the underlying molecular mechanisms?

The calcium concentration in the cytoplasm of a typical cell is .1uM. In the extracellular fluid (blood stream), it is 1mM. Therefore, there is a 10,000 fold concentration gradient across the plasm membrane. Additionally, some cellular organelles (mainly endoplasmic reticulum and mitochondria) function as a storage place with the calcium concentrations in the mM range. Cells have various calcium pumps to actively transport calcium ion across membranes against the sharp gradient at cost of ATP hydrolysis. Also, cellular proteins that are abundant and have high binding affinity to calcium, such as calmodulin, function to sequester free calcium from the cytoplasm. Consequently, the cytoplasmic calcium concentration is maintained at low levels (.1uM).

What is the cellular complex responsible for allowing proteins and other molecules to go into and out of the nucleus? Describe the main features of the gated transport controlled by these complexes.

The cellular complex responsible for allowing proteins and other molecules into and out of the nucleus is the Nuclear Pore Complex. Molecules that are less than 40-60 kD go through NPC by free diffusion. Molecules that are greater than 40-60 kD go through the NPC via active transport.

In cells, proteasome function requires ATP hydrolysis. What is the energy used for?

The energy from the ATP is used to unfold the protein.

Describe how pH plays a key role in regulating lysosomal hydrolases transport via M6P receptor?

The interaction between M6P and M6P receptor is optimal at neutral pH at TGN. There is a progressive acidification in compartment going from TGN to early then late endosome to lysosomes. At an acidic pH, M6P is dissociated from its M6P receptor. Elevating pH prevents this dissociation. The M6P receptors then become saturated with lysosomal enzymes and the cell no longer has the capacity to direct newly synthesized lysosomal enzymes to lysosomes. Instead, the enzymes are secreted from the TGN to the extracellular space by constitutive secretion.

Describe how pH plays a key role in regulating the interaction between M6P and the M6P receptor. Why does elevating endosomal pH lead to the secretion of newly synthesized lysosomal enzymes into the extracellular medium?

The interaction between M6P and M6P receptor is optimal at neutral pH at TGN. There is a progressive acidification in compartment going from TGN to early then late endosome to lysosomes. At an acidic pH, M6P is dissociated from its M6P receptor. Elevating pH prevents this dissociation. The M6P receptors then become saturated with lysosomal enzymes and the cell no longer has the capacity to direct newly synthesized lysosomal enzymes to lysosomes. Instead, the enzymes are secreted from the TGN to the extracellular space by constitutive secretion.

What is the name given to the amino acid sequence that allows the selective transport of macromolecular cargo proteins into the nucleus? Name three proteins that are required for this import and briefly describe how they function.

The nuclear import signal is the name of the amino acid sequence that allows for the import and export of proteins in and out of the nucleus. The Ran-GTPase is the directionality factor. Importin alpha is an adaptor protein. Importin beta is the import receptors.

When a protein tagged with a polyubiquitin chain is subjected to proteasome degradation, what would happen to the ubiquitins?

The polyubiquitin chain is removed by the de-ubiqutinase associated with the proteasome cap and the free ubiquitin modification of other proteins.

Describe the major reaction steps involved in production of GPI-anchored protein in the ER lumen.

The protein is transferred to the GPI anchor via the cleavage of the carboxy tail and attaching it to the inositol tail.

Describe the major steps and cellular elements involved for a protein carrying a NLS to enter the nucleus.

The protein with the NLS first gets loaded on to the nuclear import receptor which allows the protein to pass through the NPC. Once through Ran-Gef which exchanges GDP for GTP on the Nuclear import receptorl allowing for the Nuclear import receptor to exit the NPC. When exiting Ran-GAP hydrolyzes the GTP into GDP, thus allowing the Ran-GDP to dissociate from the Receptors.

Compare the similarities and differences in cellular components required for signaling to the nucleus among tyrosine receptor kinases, cytokines receptors, and serine/threonine kinase receptors.

The receptor tyrosine kinases (RTK's) have the kinase activity as part of the cytoplasmic domain of the receptor where as the cytokine receptor has a separate kinase subunit (JAKs). STATs are trascription regulators that enter into the nucleus upon phosphorylation by activated JAK's. Some RTK's can also phosphorylate certain STATs. The serine/theronine kinase receptors use smads (Co-Smads and R-Smads) to relay signals into the nucleus.

The coat complex I (COPI) activity has been shown to be critical for

The recycling of membrane proteins form the Golgi to the ER

What would you expect to happen in cells that secret large amounts of protein through the regulated secretory pathway, if the ionic conditions in the ER lumen could be changed to resemble those in the TGN?

The secretory proteins tend to aggregate in the TGN. If the ionic conditions in the ER lumen could be changed to t resemble those in the TGN, these secretory proteins would aggregate in the ER lumen. Because the aggregation is specific for secretory proteins, soluble ER proteins would be excluded from the aggregates unless the change in the ionic conditions alter the properties of these ER proteins. It is likely that such aggregates would eventually be degraded by the quality control mechanisms that operate in the ER.

The mannose 6-phosphate receptor shuttles between:

The trans-golgi network (TGN) and endosomes

Protein ubiquitination plays various roles in regulating protein activities. What are the enzymes required for protein ubiquitination? How is the substrate specificity determined in this process?

The ubiquitin-activating enzyme E1 catalyzes the formation of a covalent thirster bond with ubiquitin; the ubiquitin conjugating enzyme receives an activated ubiquitin from E1. The E3 ubiquitin ligase transfers the ubiquitin from an E2-conjugate to a target protein. The E3 enzyme confers specificity and there are roughly 500 in humans.

A transport vesicle should go from organelle "A" to organelle "B". What would happen to the vesicle if after budding from organelle "A" its Rab proteins and SNARE proteins are stripped off? Why?

The vesicle would fail to fuse with organelle B as it lacks the surface proteins (Rab GTPase and SNARE) that mediate the specific recognition between the vesicle and its destination for the fusion event.

When a polyubiquitin chain modified protein is subjected to 26S degradation, are ubiquitins digested as well? Why or why not?

Ubiquitin is not digested it is recycled and used as a signal

Describe enzymes involved in protein ubiquitination. How is the substrate specificity determined during this process?

Ubiquitination requires three types of enzyme: ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, and ubiquitin ligases, known as E1s, E2s, and E3s, respectively. The process consists of three main steps: 1. Activation: Ubiquitin is activated in a two-step reaction by an E1 ubiquitin-activating enzyme, which is dependent on ATP. The initial step involves production of a ubiquitin-adenylate intermediate. The E1 binds both ATP and ubiquitin and catalyses the acyl-adenylation of the C-terminus of the ubiquitin molecule. The second step transfers ubiquitin to an active site cysteine residue, with release of AMP. This step results in a thioester linkage between the C-terminal carboxyl group of ubiquitin and the E1 cysteine sulfhydryl group. The human genome contains two genes that produce enzymes capable of activating ubiquitin: UBA1 and UBA6. 2. Conjugation: E2 ubiquitin-conjugating enzymes catalyse the transfer of ubiquitin from E1 to the active site cysteine of the E2 via a trans(thio)esterification reaction. In order to perform this reaction, the E2 binds to both activated ubiquitin and the E1 enzyme. Humans possess 35 different E2 enzymes, whereas other eukaryotic organisms have between 16 and 35. They are characterised by their highly conserved structure, known as the ubiquitin-conjugating catalytic (UBC) fold. 3. Ligation: E3 ubiquitin ligases catalyse the final step of the ubiquitination cascade. Most commonly, they create an isopeptide bond between a lysine of the target protein and the C-terminal glycine of ubiquitin. In general, this step requires the activity of one of the hundreds of E3s. E3 enzymes function as the substrate recognition modules of the system and are capable of interaction with both E2 and substrate. Some E3 enzymes also activate the E2 enzymes. E3 enzymes possess one of two domains: the homologous to the E6-AP carboxyl terminus (HECT) domain and the really interesting new gene (RING) domain (or the closely related U-box domain). HECT domain E3s transiently bind ubiquitin in this process (an obligate thioester intermediate is formed with the active-site cysteine of the E3), whereas RING domain E3s catalyse the direct transfer from the E2 enzyme to the substrate.[58] The anaphase-promoting complex (APC) and the SCF complex (for Skp1-Cullin-F-box protein complex) are two examples of multi-subunit E3s involved in recognition and ubiquitination of specific target proteins for degradation by the proteasome. The specificity comes from the E3 protein which recognizes a specific protein, giving this process specificity.

Nitri oxide is synthesized by nitric oxide synthase from what?

arginine

Gt (Transducin) interacts with?

cGMP phosphodiesterase

t-SNARE interacts with?

v-SNARE