MCB 305 Exam 2

What types of Proteins stay synthesized in the cytoplasm?

- Cytoplasmic - Peripherial Membrane Proteins - Proteins that will go to the nucleus - Proteins that go into organelles outside the endomembrane system - Proteins that make up the cytoskeleton

Snares are innvolved in

- Docking vesicles to the target compartment. • Protein that engage in these interactions are SNAREs. • SNAREs can be divided into: v-SNAREs (incorporated into vesicles) and t-SNAREs (located in the target). - Fusion between vesicle and target membranes. • Interactions between t- and v-SNAREs pull the two lipid bilayers together.

Design or an interpret an experiment that utilizes non-hydrolysable GTP

- GTPase has different activity when bound to GTP or GDP - GEFs and GAPs catalyze switch between GDP and GTP to make the protein active or on. Non-hydrolyzable nucleotides GMP-PMP - binds in the place of GTP, but cannot be hydrolyzed to GDP therefore the protein cannot be inactive Can act as a "block" of normal GTPase activity Blocks the Sar1p GTP hydrolysis so there is no dissocaition of Sec13p and Sec23p complexes thus no vesicle fusion with the Golgi.

What are the Molecular Motors and the three types?

- Molecular motors convert energy from ATP into mechanical energy. - Molecular motors move unidirectionally along their cytoskeletal track in a stepwise manner Three categories of molecular motors: • Kinesin and dynein move along microtubule tracks. • Myosin moves along microfilament tracks

How to get Protein X from A-> B Examples of Targeting sequence or Modification on Protein X

- Phosphorylated mannose, lysosomes, KDEL -ER)

Why glycosylate?

- Promotes proper folding in the ER - Adds stability to ECM proteins in the Extracellular space - Aids some membrane protein in binding ligands on other cells (cell to cell binding)

What is Rad9 and its function

- RAD9 is one of the proteins upstream of MDM2 that regulates its activity - MDM2 is an important factor that inhibits P53 activity in normal conditions by promoting ubiquitylation and proteasomal degradation

How to get Protein X from A-> B Proteins that all recognition and fusion with the target compartments

- Rabs and Snares

What types of proteins will move to the ER to be synthesized?

- Secreted proteins - Integral Membrane Proteins - Proteins in the ER, Golgi, Lysosomes, Endosomes, and Vesicles

Role of coated pits in receptor mediated endocytosis?

- Substances that enter the cell through RME become bound to coated pits on the plasma membrane. - Clathrin-coated regions invaginate into the cytoplasm and then pinch free of the cytoplasm.

Describe the process by which ribosomes are targeted to the ER surface and proteins are synthesized?

1) A signal sequence at N-Terminus attaches to a signal recognition particle (SRP also binds ribosome) 2) SRP receptor (at surface of ER) recognizes the SRP 3) The SRP is released (requires GTP binding proteins) and the ribosomes bind to the translocon 4) Polypeptide moves into the ER cisternal space through the pore in translocon 5) Ribosome released and translocon pore closed

Describe the uptake of LDL into the cell

1) LDL binds to its receptor 2) The coated vesicle gets uncoated and allows for fusion with the endosome 3) The LDL will then be hydrolyzed in the lysosome 4) The receptor will bud off the endosome and will be transported in a vesicle to the membrane

Describe in detail the mechanism of vesicular transport between the ER and Golgi.

1) Sec12p recruits Sar1p to a bud site on the ER membrane in a reaction involving guanine nucleotide exchange. 2) Activated Sar1p (has a GTP bound) initiates the assembly of the Sec23p complex and Sec13p complex at the bud site in the production of transport vesicles. 3) GTP hydrolysis of Sar1p allows the Sec23p and Sec13p complexes to dissociate and allow for vesicle fusion with the Gogli

5 Functions of the Cytoskeleton

1) Shape - scaffold providing structural support and maintaining cell shape 2) Internal Organization - serves an internal framework to organize organelles within the cell 3) Transport - direct movement of materials within the cell (in particular vesicles and mRNA) 4) Cellular Mobility - Force generating apparatus for cellular locomotion 5) Cell Division - Facilitates changes in cell shape and the organization of chromosomes during mitosis and meiosis

Describe the pathway beginning with E2f allowing for pass from G1->S.

1) When E2f and pRb are bound on the DNA there is no gene expression 2) Cdk activation allows for pRb phosphorylation and dissociation from E2f 3) E2f will now all for transcription of mRNA coding for proteins involved in G1 to S phase ( DNA polymerase, cyclins, histones...)

Cells that crawl over a substratum display a repetitive sequence of events. What are these events?

1. Protrusion of leading edge/lamellipodium 2. Gripping of surface at lamellipodium (integrin) 3. Contractile forces (using actin/myosin) pull cell body forward 4. Rear of cell released from substratum and pulled forward

Cyclin-Cdk is activated and inactivated in what forms and what enzymes are responsible?

ACTIVE: Cdk-Cyclin are phosphorylated (1 P) to inactivate it Wee1 kinase will come in and add a second P making it inactive. However Cdc25 phosphatase can come in and take away the 2 P and make it active again

When DNA is damaged by ionizing radiation

ATM detects the damage, p53 is stabilized and phosphorylated, the cell cycle is arrested

Describe the basic structure of actin filaments, including the mechanism behind the tread milling behavior

Actin • Microfilaments are composed of actin and are involved in cell motility. •Using ATP, actin polymerizes to form actin filaments ("F-actin"). • Filaments form from monomers • A small filament (seed) must be "nucleated" first • Minus end is shrinking, while plus end is growing • The steady state of this growing/ shrinking is called "treadmilling"

Anterograde vs. Retrograde

Anterograde is going forward (Nucleus to ER cis to trans Golgi to Plasma Membrane) Retrograde is the opposite of going backwards

How does glycosylation in the Golgi occur?

As the protein goes from cis to trans each cisternae there are enzymes within cisternae that allows for modification

The Signal Hypothesis

Blöbel, Sabatini and Dobberstein proposed that the site of protein synthesis is determined by information contained in the N-terminal portion of the protein, the first part to emerge from the ribosome.

Difference between Vesicle Budding vs Vesicle Fusion

Both are used with Transport vesicles. Budding is when receptors on the donor compartment have molecules bind and cause budding and then a vesicle forms and then on the recipient compartment there is fusion.

CAK adds Wee 1 adds Cdc25 removes

CAK - adds Thr 161 -P (activating P) Wee1 - adds Tyr 15 -P (inhibitory P) Cdc25 - removes Tyr 15-P (inhibitory P)

Difference between COPII and COPI

COPII goes from ER to the cis-Golgi COPI goes from cis-Golgi to the ER

What is the centrosome and centriole?

Centrosome - structures responsible for initiating microtubules in animal cells. • It contains two barrel shaped centrioles surrounded by pericentriolar material (PCM). • Centrioles are usually found in pairs

How to get Protein X from A-> B Proteins to selectively package proteins into vesicles

Coat proteins and adaptor proteins

Describe the organization of a coated vesicle

Coated vesicles also contain adaptors between clathrin and membrane. • AP2 adaptors engage the cytoplasmic tails of membrane receptors (which carry cargo). • AP2 adaptors also interact with Clathrin

MPF =

Cyclin + CDC2

Why do cyclin levels fluctuate during the cell cycle?

Cyclin levels fluctuate during cell cycle and serve as regulators of entry and exit from different stages As we enter the next stage of the cycle the previous cyclin (cyclin E) will be degraded and Cyclin A will be expressed

Function of Cyclins and Cdks

Cyclins - regulatory proteins that are made and degraded Throughout the cell cycle Cyclin dependent kinases (Cdk) - Regulatory proteins that (when bound to cyclins) are able to phosphorylate downstream targets

Function of Dynein and the direction it moves?

Dynein - responsible for the movement of cilia and flagella. Moves from + to -

Based on this model for the function of pRb, what do you predict would happen in a cell where both copies of the Rb gene were mutated and the Rb protein was non-functional?

E2F would promote gene activation which would promote the G1-S transition and cellular proliferation

The KDEL receptor is responsible for recognizing an ER retention signal on ER proteins. Which of the following would be most likely if yeast cells contained a mutation that made the KDEL receptor non-functional?

ER enzymes would be secreted from the cell rather than stored in the ER.

Describe the early simplistic model, vesicular transport model and cisternal maturation model of the Endomembrane system

Early simplistic Cisternal Maturation Model: Vesicular Transport Model: Cisternal Maturation Model:

What is Endocytosis, pinocytosis, and receptor-mediated endocytosis

Endocytosis - uptake of cell surface receptors and bound extracellular ligands. - Pinocytosis - nonspecific uptake of extracellular fluids. - Receptor-mediated endocytosis - uptake of specific extracellular ligands following their binding to receptors.

Mannose-6-phosphate is an example of what

Example of a Zip Code Protein modification in the Golgi - Adds the phosphate in the cis-Golgi and allows to bind to receptor to target to late endosome/lysosome via the receptor

KDEL is an example of what?

Example of zip code • Resident proteins of the ER contain an amino acid sequence at the C-terminus serving as a retrieval signal. • Specific receptors capture the molecules and bring them to the ER in COPI-coated vesicles. • Each membrane compartment may have its own retrieval signals.

What are motor proteins

Force generating proteins that move along cytoskeleton • Involved in: directed movement of vesicles, organelles, mRNA and some proteins inside cells • Involved in muscle contraction and mitosis

The Underlying Basis of Microtubule Dynamics

GTP is required for microtubule assembly. - Hydrolysis of GTP leads to a replacement of bound GDP by new GTP to "recharge" the tubulin dimer.

Desribe gap and tight junctions

Gap junctions - sites between animal cells for intercellular communication. • Composed entirely of membrane protein connexin. • Connexins are organized into a complex called connexon Gap Junctions Mediating Intercellular Communication • Gap-junction intercellular communication allows the passage of lowweight molecules. • Gap junctions can allow integration of activities of individual cells into a functional unit (for example ions in cardiac muscle). • Relatively little selectivity Tight junctions seal the extracellular space between cells • Claudins form the major structural component of TJs

Describe Dynamic Instabiltiy

Growing and shrinking microtubules can coexist in the same region of a cell. - A given microtubule can switch back and forth between growing and shortening phases. - It is an inherent property of the plus end of the microtubule. - Proteins called +TIPS regulate the rate of growth and shrinkage.

Describe the role of HDLs vs LDLs

High-density lipoproteins (HDLs) transport cholesterol from tissues to the liver for excretion. - HDLs are associated with lowering cholesterol levels whereas LDLs are associated with high blood cholesterol. - Deposition of LDL leads to plaque formation on the inner walls of blood vessels.

Why is retinoblastoma more common in individuals who have inherited the RB deletion and have the genetic predisposition for retinoblastoma?

If the RB gene is already mutated or deleted on one of the homologous chromosomes, only one hit rather than two is needed to cause development of retinoblastoma; thus the probability is higher.

What are the actions of drugs on Synapses

Interference with the destruction or reuptake of neurotransmitters can have dramatic physiological and behavioral effects. - For example, antidepressants (Prozac, Zoloft: called Selective serotonin reuptake inhibitors) block the serotonin uptake receptor - Cocaine and methamphetamine have a similar, less selective, mode of action

Describe what are LDLs and what happens to them in cells

Low-density lipoproteins (LDLs) are a complex of cholesterol and proteins. - LDL receptors are transported to the plasma membrane and bound to a coated pit. - LDLs are taken up by RME and taken to the lysosomes, releasing the cholesterol for use by the cells. - Even though coated pits take up only about 2% of plasma membrane they contain 50- 80% of LDL receptors

What regulates the stability of p53?

MDM2 is an important factor that inhibits P53 activity in normal conditions by promoting ubiquitylation and proteasomal degradation • Ubiquitation: a "tag" is added to p53 to signal it for destruction • Phosphorylation of p53 prevents interaction with MDM2, protecting it from degradation and allowing activation of cell cycle arrest

Definition of microsome, protease, mature light chain, pre-light chain, and globin

Microsome - cell free ER (contain ER components on the membrane and inside), large vesicle-like pieces of the ER Protease - destroys proteins that are not protected inside the microsomes Mature light chain - IgG molecules same size as what is normally secreted from the cell Pre-light chain - (smaller molecule that you get if you make IgG in the cell free systems with ribosomes but not ER) Globin - cytoplasmic protein

Structure of microtubules

Microtubules are hollow, cylindrical structures The microtubule is a set of globular proteins arranged in longitudinal rows called protofilaments. - Microtubules contain 13 protofilaments. - Each protofilament is assembled from dimers of α- and ß-tubulin subunits assembled into tubules with plus and minus ends.

Function of Microtubles

Microtubules as Structural Supports and Organizers - The distribution of microtubules determines the shape of the cell. - Microtubules maintain the internal organization of cells.

Functions of microtubules in mitosis

Microtubules provide the force and the spatial organization to align and separate the chromosomes into the two daughter cells

p53 description

Most commonly mutated gene in human cancers • About half of human cancers contain cells with mutations in both copies of p53. • P53 is a transcription factor that activates genes involved in control of the cell cycle and apoptosis • P53 has many roles in controlling the cell cycle - Unstable and usually degraded in the cell (no P) but when P it is stable and cause for transcription of p21 protein which will inactivate cdk and arrest the cell cycle

Describe the Endomembrane system

Organelles of the endomembrane system are part of an integrated system where materials are shuttled back and forth - Materials are shuttled between organelles in membrane bound transport vesicles - Upon reaching their destination the vesicles fuse with the membrane of the accepter compartment

Permissive vs Restrictive Mutants

Permissive Temperature - phenotype "normal" • Restrictive Temperature - phenotype "abnormal" • Typically due to a slightly misfolded protein that is functional at permissive temp but not at restrictive temp.

What effect does the binding of the SRP to the growing polypeptide chain and the ribosome have on protein synthesis?

Protein synthesis ceases temporarily.

Proto-oncogenes

Proto-oncogenes genes encode proteins that promote cell growth. • Mutations in one copy of the gene (which is then called an oncogene) can support cancer progression • Oncogenes may develop in different ways, but in general they create a protein that has escaped its normal regulation (either regulation of the protein function or regulation of how much of the protein is made or when it is made)

Which of the following BEST describes the role of COP proteins in the endomembrane system?

Serve as coat proteins helping to promote vesicle formation

Cdk activation requires?

The Cdk is inactive and then cylcin will bind and then the CAK (cyclin acitvating kinase) will come in and fully activae the Cdk-cyclin

Describe the Golgi Complex

The golgi complex is a stack of flatted cisternae, it is divided into several functionally distinct compartments, and the cis face of the golgi faces the ER and the trans face is on the opposite of the stack.

What is the directionality of a proteins from the ER, through the endomembrane systems, to the surface of the plasma membrane

The protein it going to made facing the lumen of the ER, vesicle and golgi and when it is done it will face the outside of the cell.

What allows smooth and rough vesicles (microsomes) to be readily separated by density gradient centrifugation?

Their differences in density

What happened to COPI-coated vesicles within the cell when the cell was treated with GTP analogues that could not be hydrolyzed?

They accumulated in the cytoplasm

What is the function of chaperone proteins

They regulate folding of new proteins in the ER

Function of Rabs

They target vesicles to a particular compartment by tethering vesicles to the target compartment. • Process is mediated by G proteins called Rabs. • Rabs play a key role in vesicle targeting by recruiting specific tethering proteins.

When a microtubule is growing, the plus end is present as an open sheet to which GTP-dimers are added. A cap of GTP-dimers can often form on the growing microtubules during rapid growth periods. How does this cap form?

Tubulin dimers are added to the microtubule faster than the GTP is hydrolyzed

Tublin is a GTPase and how does this affect micro-instability

Tubulin heterodimers • GTP-tubulin added at + end • GTP ends more stable • Tubulin is a GTPase • "Catastrophe" when cap is lost and GDP is bound

Tumor suppressor genes

Tumor-suppressor genes encode proteins that restrain cell growth. • Mutations in both copies of the gene for these proteins are needed to support cancer progression

Function of Kinesins and the direction they walk?

Two motor heads each with a catalytic core 1) When one of the motor heads binds to the microtubules the ADP to be released from head and ATP will rapidly binds to the head. 2) this will cause the back head (With ADP) bound to be thrown in front of the ATP and brings it near the next binding site on the microtubules. 3) The trailing head with the ATP will hydrolyze ATP to ADP and the process reoccurs Moves from - to + whiles some members move from + to -

How does drug Taxol Work?

Used to treat ovarian and breast cancer as well as certain lung cancers. Taxol stabilizes microtubles, destroying the normal dynamic instability balance - Cells treated with Taxol before Mitosis, fail to perform normal bi-polar spindle and do not separate chromosomes normally.

A graduate student in your lab has created a yeast strain that has a mutant form of Sar1p. She purified this mutant protein and showed that it could bind GTP, but could not hydrolyze it. You suggest that she compare the protein from her mutant to the protein from wild-type cells in a vesicle budding and fusion experiment (like the one we examined in class). What results should you expect to see?

Vesicle budding and fusion will occur with the wild-type protein, but only budding will occur at a significant level with the mutant protein.

Describe in general the mechanism of vesicular transport between the ER and Golgi.

Vesicular Transport • Materials are carried between compartment using coated vesicles. • Protein coats have two distinct functions: 1) Cause the membrane to curve and form a vesicle. 2) Select the components to be carried by vesicle

A "catastrophy" happens:

When tubulin throughout an entire microtubule is bound to GDP, instead of GTP

Based on this model, which of the following do you predict would happen if microtubules were formed in vitro in the presence of GTP or GMPCPP

With GMPCPP, microtubules will grow at the same rate, but will have fewer "catastrophes".

What causes the rapid drop in Cdk activity that leads to the exit from mitosis and the entry into G1 typically exhibited by cells as they finish division?

a plunge in mitotic cyclin concentration

Entry into mitosis is triggered by?

by activation of maturation promoting factor (MPF). • MPF consists of : cyclin (a regulatory subunit) and a cyclin dependent kinase (cdk). • The cyclin levels fluctuate predictably during the cell cycle. • Increased concentration of cyclin activates the kinase

pRB controls what?

controls transcription of genes required for cell cycle progression IT IS A TUMOR SUPPRESSOR

What are the two sites within a cell at which protein synthesis is generally thought to occur?

cytosolic surface of RER and free ribosome

Clathrin-Mediated Endocytosis found to be important in disease - What disease and what happens?

ells from individuals with familial hypercholesterolemia (FH) are unable to regulate cholesterol biosynthesis in response to LDL.

LDL receptors are found mostly on the outer surface of cells even in the absence of ligand. Where specifically on the cell surface are they found?

in the coated pits

The regulatory subunit of maturation-promoting factor ________.

is called cyclin because its concentration rises and falls predictably as the cell cycle progresses

What is "ncd" homologous too?

ncd is homologous to conventional kinesin moves from + to - THOUGH

The development of RB requires

requires both copies of RB to be altered or eliminated.

What the cisternal maturation model

suggests that the Golgi cisternae are transient structures that form at the cis face of the stack by fusion of membranous carriers from the ER and ERGIC and that each cisterna travels through the Golgi complex from the cis to the trans end of the stack, changing in composition as it progresses?

What molecules do the AP2 adaptors of the clathrin coat connect?

the cytoplasmic tails of specific membrane receptors and clathrin molecules

Progression through the cell cycles requires?

the phosphorylation and dephosphorylation of critical cdk residues

Compare and contrast the ways in which actin contributes to force generation, via coupling with myosin or via branching

• All myosins share a characteristic motor head for binding actin and hydrolyzing ATP Myosins can be divided into two groups: • Conventional (type II) myosins (includes those abundant in muscle tissue) • Unconventional myosins • Myosins are a family of motor proteins that move along actin filaments • Myosin pulls actin filaments together to produce the force needed for cytokinesis (the contractile ring)