Cell Bio Midterm Study

How are monomers joined together? (what type of reaction).

A condensation reaction creates bonds between monomers, releases H20.

What is a histone octamer composed of?

A histone octamer is composed of 2 of each type of histone: H2A, H2B, H3, H4. Each histone has a protein tail that protrudes from the octameric core.

How are monomers taken apart?

A hydrolysis reaction breaks bonds between monomers by addition of H20.

What is a nuclear localization signal (NLS)? How does that become attached to a protein?

A nuclear localization signal is an amino acid sequence that 'tags' a protein for import into the cell nucleus by nuclear transport.

What is peripheral heterochromatin, and what is its relevance to the human genetic disorder progeria?

A peripheral heterochromatin is a layer of dense heterochromatin found at the periphery of the nucleus. The nuclear lamina is associated with progeria. Lamins are important filaments largely making up nuclear lamina. Mutations in lamin genes cause premature aging syndromes. Peripheral heterochromatin disappears with age and that brings up the question of whether or not loss of association of chromatin with the nuclear lamina causes cell aging.

Describe why misfolded prion proteins are toxic. Related: Why are disease-related amyloid fibrils so structurally stable?

A single misfolded prion goes on to induce the same folding effect in all the other (initially normal) prion proteins. Disease-related amyloid fibrils are so stable because they form a double beta sheet that is highly resistant to protein degradation and it'll build up in the extracellular matrix.

How does a synthetic trans-fat compare against naturally unsaturated and saturated fats?

A synthetic trans-fat is hydrogenated meanwhile, naturally unsaturated and saturated fats are not hydrogenated

How do amphipathic alpha helices embed within a lipid bilayer?

All polar amino acids are on one side of the helix and all non polar amino acids are on the other side of the helix. The hydrophobic side will embed in the middle of the bilayer and the hydrophilic side will remain in contact with aqueous solution.

Review: What is an amphipathic alpha helix? How does this differ from a hydrophobic helix?

An amphipathic alpha helix has hydrophilic amino acid side chains on one side and hydrophobic amino acids side chains on the other side. This amphipathic alpha helix of Sar-1 make it embed in one side of the lipid bilayer (ER membrane).

Describe the general stepwise process of N-linked glycosylation.

Before an oligosaccharide can be attached to a protein, first the oligosaccharide has to be assembled and that process is started on the cytosolic side of the ER membrane. The oligosaccharide is assembled onto a carrier which is inserted into the ER membrane. The growing oligosaccharide is translocated from the cytosolic side of the ER membrane to the ER lumen which is catalyzed by a flippase. Once the assembly of the oligosaccharide is done it is transferred and attached to a protein at the nitrogen atom of one of its amino acids to make a glycoprotein. After, the glycoprotein is moved into the Golgi apparatus for further modifications. Monosaccharides can be removed or added.

What determines whether amphipathic molecules form a bilayer or a micelle?

Bilayer: Small polar headgroup with multiple nonpolar fatty acid tails. Micelle: Big polar headgroup and single nonpolar fatty acid tails.

How difficult is it, comparably, to break each of these types of bonds?

Bond strength (Easiest -> Hardest): Hydrogen, Ionic, Polar Covalent, Nonpolar Covalent.

How is carbohydrate synthesis different from protein and DNA synthesis?

Carbohydrates: No template Happens in different steps Requires different enzyme at each step Primary structure can be linear or branched DNA: Uses template Happens in repeated series of identical steps Same enzyme, or set of enzymes Primary structure is linear

What are the 4 important macromolecules of biological systems, and what are the building blocks of each?

Carbohydrates: Monosaccharides Lipids: Glycerols + Fatty Acids Proteins: Amino Acids Nucleic Acids: Nucleotides

If you dissolved a spoonful of salt (NaCl) in a glass of water, be able to define what the cation and anion are.

Cation: Sodium Anion: Chlorine

How do hibernating animals compensate for the effect of their internal temperature change on their cell membranes?

Cells compensate by increasing the incorporation of unsaturated fatty acids into their cell membranes.

Compare and contrast channel and transporter proteins. What is a "pump" membrane transport protein?

Channel proteins: form hydrophilic pores across membranes through which certain substances pass through by diffusion. Passage depends on size and electric charge of the solute. Transporter proteins: change shape to pass solute to the other side of the membrane. Passage depends on the solute's fit into a specific binding site on the transporter.

What is a chimeric gene, and how is it useful?

Chimeric genes form through the combination of portions of two or more coding sequences to produce new genes.

Compare and contrast clathrin- and caveolin-mediated endocytosis.

Clathrin-mediated endocytosis: Bud off the membrane by recruiting proteins to the surface of the membrane that help the membrane curve. Caveolin-mediated endocytosis: Is a co-protein that binds cholesterol. Caveolin proteins are thought to be associated with lipid rafts which are known to be rich with cholesterol. Caveolins are not transmembrane proteins but rather they insert a hydrophobic loop of protein inside the lipid bilayer. Caveolin are thought to bud off the membrane by controlling the lipid composition of the caveolin membrane rather than by the assembly of the cytosolic protein code. Similarly to clathrin-mediated endocytosis, caveolin are pulled off the membrane by dynamin.

What determines whether a protein will be translated in the cytosol or on the ER surface?

Co-translational import: Ribosome synthesizing polypeptides that will be exported from the cell. These ribosomes become attached to the ER membrane early in translation and polypeptide chains are transferred across the membrane as synthesis takes place. Post-translational import: Employed for polypeptides that will either stay in the cytosol or wind up in mitochondria, chloroplasts, peroxisomes, or the interior of the nucleus. After translation of polypeptides coming from these ribosomes is complete, they are released from the ribosomes and they remain in the cytosol or are taken up by the appropriate organelles.

Define concentration gradients, electrochemical gradients, and how they relate to passive vs. active transport

Concentration gradient: the magnitude of difference in the concentration of a solute on the opposite side of the membrane. Electrochemical gradient: Net force driving a charged solute across a membrane = composite of concentration gradient and membrane potential across membrane. Passive transport: allows solutes to move down their concentration gradients. Occurs spontaneously and requires no energy input. Active transport: moves solutes against their concentration gradients, and requires input of energy.

What sort of content can easily pass through a solid bilayer?

Content that can easily pass through a solid bilayer is a small molecule that is hydrophobic and nonpolar. Ex. Hydrophobic molecules: O2, CO2, N2, steroids, hormones.

What is the role of the CopII coat proteins?

CopII coated vesicles help transport cargo from the ER to the Golgi apparatus.

Define a covalent bond vs. an ionic bond vs. a hydrogen bond.

Covalent bond: Bonds between atoms that share electrons. Ionic bond: Electrons are transferred from one atom to another. Hydrogen bond: Weak type of bond that forms between a hydrogen that is covalently bonded to one molecule (and is therefore positively charged) and a nearby atom that is negatively charged.

What is the central dogma's key players?

DNA replication (DNA polymerase): DNA to DNA Transcription (RNA Polymerase): DNA to RNA Translation (Ribosome): RNA to Protein

How does the dynamin protein help to detach a vesicle from the plasma membrane?

Dynamin is an ATP-powered protein that pinches off the new vesicle by squeezing the water out so bilayer areas can fuse together.

What is an "R-group," and how does it relate to how amino acids are classified?

Each of the 20 amino acids has a specific side chain, known as an R group. This allows amino acids to be grouped according to the chemical properties of their side chains.

What is the difference between a polar vs. a non-polar covalent bond, and how do you identify each?

Electronegativity: the power of an atom in a molecule to attract electrons to itself Polar covalent bonds: Big differences in electronegativity. Nonpolar covalent bonds: Small differences in electronegativity.

Explain 3 different ways that the nuclear lamina (or proteins associated with the nuclear lamina) can suppress gene expression.

Emerin proteins that are part of the nuclear lamina bind to the chromatin and help to pack it and specify it as heterochromatin. Emerin binds to histone deacetylase which increases the enzymes activity more than two-fold, thus resulting in more densely packed chromatin. Emerin binds to transcription factors which is associated with the repression of expression of genes that those transcription factors usually activate in the euchromatin.

How are proteins removed from the nucleus by Exportin?

Exportin binds to proteins that have a nuclear export signal (NES). Ran-GTP activates exportin, which then picks up an NES cargo. Exportin associates with unstructured (FG) nucleoporins to transit the pore. Ran-GDP (in cytoplasm) hydrolyzes to Ran-GTP. Ran-GDP and NES cargo dissociate from exportin.

What is the FRAP technique, and how did it support the fluid mosaic model?

FRAP is a method for determining the kinetics of diffusion through tissue or cells.

Why and how does the fatty acid content of a lipid bilayer affect its fluidity?

Fatty acid length: Standard length: Becomes easier to freeze and increases the tendency of tails to interact with each other, thus decreasing fluidity. Shorter length: Becomes harder to freeze and decreases the tendency of tails to interact with each other, thus increasing fluidity.

Consider examples of folding, barrier, structural support, identification

Folding: Oligosaccharides on a protein are required in order for that protein to be transported to the golgi apparatus for further processing so that it can be modified into its final form. Improper pattern of oligosaccharides on a protein would make that protein translocated back to the cytosol where it would be ubiquitous and degraded in a proteasome. They make folding intermediates more soluble, preventing their aggregation and they also can help mediate the binding to chaperones. Barrier: Oligosaccharides tend to make a glycoprotein more resistant to digestion by proteolytic enzymes. An example of a barrier are lung and intestinal cells that are surrounded by a mucous coat. Mucins are a type of glycoprotein and that sugar coat protects those cells against many pathogens. Structural Support: Glycoproteins in the extracellular space are structurally strong. An example of that is collagen, one of the main structural proteins in the extracellular matrix. Polysaccharide chains are strongly hydrophilic which tends to gel and they also have a high density of negative charges which attracts sodium ions, causing water to be sucked into the matrix creating a swelling pressure turgor that enables the matrix to withstand a lot of force. The cartilage matrix that lines the knee joint can support pressures of hundreds of atmospheres. Identification: Lysosomal hydrolase (enzyme) gets decorated with the oligosaccharide M6P. This tag binds to a M6P receptor in the trans-Golgi. The M6P receptor is ultimately recruited into lysosome- directed vesicles. Sugar chains differentiate your blood types and help differentiate cells that are sick and that are healthy.

What is a GTPase, and how are Rab proteins an example of that? How are GEF and GAP proteins involved?

GTPase are proteins that bind and hydrolyze GTP (guanosine triphosphate) to GDP (guanosine diphosphate) and are important regulators of cell activity. RAB is a GTPase. When RAB is bound to GTP it is in its active form and can bind to effector proteins. RAB is inactivated by GAP proteins which stimulate RAB to hydrolyze its GTP, removing a phosphate group and turning GTP to GDP. When RAB is bound now to a GDP molecule it is inactive, so RAB cannot bind an effector protein and therefore cannot help a vesicle dock to a membrane. A GEF molecule can activate RAB by binding to it and causing it to release the GDP molecule and open up its nucleotide binding site so that a new GTP can be bound and make the RAB protein active again.

What is a GTPase, and how is the activity of a GTPase generally controlled?

GTPase are proteins that bind and hydrolyze GTP (guanosine triphosphate) to GDP (guanosine diphosphate) and are important regulators of cell activity. They are subject to control by standard regulatory elements.

How is human health impacted by glycosylation? Why did an understanding of glycosylation enable blood transfusions to successfully occur for the first time? Future cancer treatments may rely on understanding glycosylation -- why?

Glycosylation enabled blood transfusions to successfully occur for the first time because it allows for the determination of an individual's blood type to make sure they get the correct blood transfusion since our bodies need the correct sugars. Cancer has the ability to express new sugars which mesmerize the immune system. Scientists know the mechanism enabling them to create new medicines that wake up the immune cells and tell them to ignore the sugar and go after the cancer cells.

Why are proteins glycosylated? What benefits are conferred by that?

Glycosylation is an effective way of generating diversity in proteins and modulating their properties due to the inherent structural variations of glycans.

What does it mean for a pump to be "gradient-driven" vs. "ATP-driven"?

Gradient-driven: harness the movement of one solute along its electrochemical gradient to transport another solute against its electrochemical gradient. ATP-driven: use the energy released by the hydrolysis of ATP to drive the transport of a solute against its electrochemical gradient across the membrane.

How can histone tails be modified? What enzymes do those modifications? What are the consequences on chromatin state and gene expression with each type of modification? (i.e. acetylation, phosphorylation, methylation)

Histone tails can be modified to impact the DNA they are bound to by altering the amino acids making up the histone tail by covalently bonding chemical groups to them to give them a more positive or negative charge. Acetylation: If a histone tail contains a lysine, that lysine can be acetylated by an enzyme called histone acetyl-transferase (HAT) which removes two hydrogens from the nitrogen that is carrying the positive charge on the lysine and is replaced by an acetyl group. That effectively reduces the positive charge on the lysine residue, thus the histone tail will be less attracted to DNA. This modification can go in reverse through the help of histone deacetylase (HDAC) which removes an acetyl group from lysine and makes it positively charged, and therefore more attracted to DNA. Phosphorylation: If a histone tail contains a serine, that serine can be phosphorylated by an enzyme called kinase which adds phosphate groups in a reaction that requires ATP, making it less attracted to DNA. This modification can be reversed by an enzyme called phosphatase which removes the phosphate group from the serine and makes it more attracted to DNA. Methylation: If a histone tail contains a lysine, that lysine can be methylated by an enzyme called methyl-transferase which adds methyl groups. This modification can be reversed by an enzyme called demethylase which removes the methyl group from the lysine. Instead of methylation making an amino acid more positive or negative it creates a signal that other proteins can read so the particular type of amino acid being methylated, the number of methyl groups added, and the particular histone being modified. Denser packing with DNA: H3K9me3 and H3K27me3 Looser packing with DNA: H3K4me2 and H3K4me3

What is homeoviscous adaptation?

Homeovisvous adaptation: The capability of some organisms to regulate membrane fluidity by altering membrane lipid composition.

What is an example of how Hsp70-like proteins affect existing interactions between proteins?

Hsp70-like proteins are called upon by the cell to take action in stressful conditions for the cell. An example is heat shock proteins that help the cell when temperatures are high since high temperatures can denature proteins.

How does Hsp70-mediate correct protein folding? Describe each step.

Hsp70-mediate binds temporarily to the hydrophobic regions of the polypeptide as the polypeptide is being made. This process requires energy from ATP. Hsp70-mediate first loosely binds a polypeptide while it's being translated coming out fresh from the ribosome. It uses ATP hydrolysis to bind more tightly to the polypeptide. Hsp70-mediate then acquires a new ATP molecule and falls off after it has done its job.

Rank how easily hydrophobic molecules, small/large uncharged polar molecules, and ions diffuse through lipid bilayers?

Hydrophobic: O2, CO2, N2, steroids, hormones. Small uncharged polar molecules: H2O, urea, glycerol. Large uncharged polar molecules: glucose and sucrose. Ions: H+, Na+, HCO3-, K+, Ca2+, Cl-, Mg2+

How does GTP-Ran control the directionality of import by Importin proteins?

Importin recognizes a NLS on a cargo protein in the cytosol and binds to it. The complex they form moves into the nucleus through the nuclear pore and once it arrives in the nucleus GTP-Ran interacts with importin and causes the NLS containing cargo protein to be released into the nucleus. Once this release occurs GTP-Ran and importin complex is released into the cytosol where GTP-Ran is hydrolyzed to become GDP-Ran which enables it to be released from importin.

Where/how is Sar-1 activated? How does active Sar1 contribute to CopII coat formation?

In its active state Sar-1 is associated with the ER membrane and helps assemble a CopII coat. The GEF for Sar-1 is only found in the ER so when that GEF binds to an inactive Sar-1 GDP complex it triggers the release of the GDP molecule causing a conformational change in Sar-1. This allows Sar-1 to be in its active state and bind to the membrane and what the conformational change actually is is an exposure of an amphipathic alpha helix protein within it. The GTPases that regulate the assembly of CopII vesicles is called Sar-1.

Can you describe integral, peripheral and lipid-anchored membrane proteins? (What distinguishes these types from each other?)

Integral proteins: contain hydrophobic segments which physically embeds them in the bilayer. Peripheral proteins: associated via non-covalent bonds with integral proteins. Lipid-anchored proteins: covalently attached fatty acid holds protein at bilayer.

How does ER shape respond to cell cycle timing, and what are the implications of that?

Interphase: often shows sheet-like (rough) ER. Mitosis: ER generally shifts to tubular (smooth). Implication: ER-based translation stops (mostly) during mitosis.

Describe the basic properties of an ion channel. What does it mean for an ion channel to be "gated" and what can control whether the gate is open or closed?

Ion-selective: ion channels are lined with charged amino acids which only bind to specific ions. Ion-gated: ion channels are closed and only open by different stimuli.

Explain the central dogma

It is the process by which instructions in DNA is converted into a functional product

What is the function of a karyopherin? How do they aid protein transit through a nuclear pore?

Karyopherin proteins enable moving target proteins in and out of the nucleus. Big cargo has NLS and is bound to Karyopherin. Karyopherins bind to the FG repeats in the nucleoporin proteins. Movement of nucleoporins can carry the Karyopherin/cargo complex across the pore.

Outline the steps of clathrin-mediated endocytosis. Identify the proteins involved in clathrin-mediated endocytosis, and evaluate what would happen in their absence or if they underwent a conformational change.

LDL receptor in cell membrane binds to an LDL particle. (The Receptor is a single pass, alpha helical transmembrane protein.) Receptors bind to Adaptins. (Adaptins are also bound to Clathrin.) Clusters of Clathrin form round baskets, containing LDL-bound receptors. Dynamin pinches off the "neck" of the membrane to separate a new clathrin-coated vesicle away from the plasma membrane. Vesicle uncoated (also requires energy) and is called "early endosome". Early endosomes fuse with the "sorting endosome." Recycling endosome sends LDL Receptor back to the plasma membrane. Cholesterol is directed to the lysosome.

Where are lemon juice and bleach, respectively, on the pH scale? Why?

Lemon juice: 2.3 because it has an excess of H+ which is acidic. Bleach: 12.6 because it has an excess of OH- which are basic.

How/why does excitation light make a fluorophore glow?

Light hits the fluorophore molecule (this = excitation light) which causes one electron in the fluorophore to jump into a higher energy orbital than normal. When that electron falls back into its orbital, the fluorophore emits light of a longer wavelength (this = emission light)

Why can't a linear/unfolded protein reach the whole way through a lipid bilayer?

Linear/unfolded proteins cannot reach the whole way through a lipid bilayer because the peptide bonds are inherently polar.

How do lipid rafts contribute to endocytosis?

Lipid rafts are plasma membrane microdomains enriched in cholesterol and sphingolipids that are involved in the lateral compartmentalization of molecules at the cell surface. Internalization of ligands and receptors by these domains occurs via a process defined as raft-dependent endocytosis.

What are the main players in the ubiquitin-proteasome system (UPS)? What is the function of each component, and how do they work together?

Main players: E1: the Ubiquitin-activating enzyme (teal blue) E2: the Ubiquitin-conjugating enzyme (dark blue) E3: the Ubiquitin Ligase (medium blue) Pathway: Ubiquitin is conjugated to the Ubiquitin-activating enzyme (E1) Ubiquitin is transferred to Ubiquitin-conjugating enzyme (E2) Note: E2 is bound to E3 in a complex The E3 subunit of the complex binds to a protein to be degraded. This brings the protein target close to E2 and the Ubiquitin tag. E2 transfers the ubiquitin to a lysine residue in the target protein. E2 and E3 dissociate from the ubiquitin-tagged protein and from each other The whole cycle repeats to create a poly-ubiquitin chain. This poly-ubiquitin chain is needed to target a protein for degradation (= what the proteasome detects)

How do vesicles fuse with other bilayers (destination compartments) in a cell? Describe the role of Rabs/Rab effectors and SNARE proteins in that process.

Membrane proteins drive membranes to fuse with the right target. Rab protein docks with its effector on the target membrane. v-SNARES interact with proteins on the target membrane (t-SNAREs)

What is the "indirect immunofluorescence" technique?

Method for optical detection of proteins. Uses antibodies created in other animals to attach to specific proteins. These antibodies are tagged with a fluorophore that will be visible under a microscope.

How does Hsp60 enable protein refolding? Describe each step.

Misfolded protein has (pink) nonpolar domains exposed. Misfolded protein associates with opening of Hsp60 chamber (pink domains are grabbed by hydrophobic protein binding sites). A cap of proteins (GroES) binds and isolates the protein in the chamber. Chamber stretches upward, exposing Hsp60 hydrophilic residues to the target protein. This pushes nonpolar domains of target protein back inward, driving re-folding of the target protein. After ~15 seconds, ATP hydrolyzes, and target protein is released.

Describe how 3 transport proteins work together in a gut epithelial cell to use glucose from food to power the body

Na+/Glucose symporter takes up glucose from the gut lumen by harnessing that energy from the sodium ion gradient across the portion of the membrane that is exposed to the lumen. Na+/K+ antiporter is constantly moving sodium out of the cell because in order for the Na+/Glucose symporter to function there needs to be a higher concentration of sodium ions in the gut lumen than inside the cell. Glucose uniporter moves glucose along its concentration gradient so if there is a lot of glucose building up in the epithelial cell it is going to get moved out into the extracellular fluid.

How does modifying histone charge affect 1) DNA affinity for the octamer, 2) chromatin compaction, and 3) gene expression? What is the relationship between chromatin compaction and gene expression?

Negative charge: Lower affinity of histone tails for DNA = looser packaging = more gene expression Positive charge: Higher affinity of histone tails for DNA = tighter packaging = less gene expression Density of DNA packaging affects gene expression: DNA that is very loosely packed in the nucleus, RNA polymerase can access DNA easily to transcribe mRNA from genes. Thus, there will be more gene expression from euchromatin. DNA that is very tightly packed in the nucleus, RNA polymerase cannot access DNA to transcribe mRNA from genes. Thus, there will be low/no mRNA expression for heterochromatin.

In a folded protein, where are most of the nonpolar vs. polar residues located? Why?

Nonpolar amino acid residues are located in the interior of the molecule to stabilize the structure and hydrophilic residues are located on the molecular surface to make capable interaction with water.

What is the structural difference between normal and misfolded prion proteins?

Normal prion protein: Alpha helical containing structure. Misfolded prion protein: Beta sheet containing structure.

What is a nucleosome composed of?

Nucleosome is the first level of DNA compaction. It contains histone proteins which are small basic (positively charged) highly conserved proteins. Nucleosome "bead" is composed of 8 histone molecules and 146 base pairs of DNA.

Compare and contrast O-linked vs N-linked glycosylation. What atom is the sugar bonded to for each? What types of glycosylation occur in ER vs. Golgi?

O-linked glycosylation: When the sugar group has been bonded to an oxygen atom typically the hydroxyl oxygen in the amino acids serine and threonine. Only occurs in the Golgi apparatus. N-linked glycosylation: When the sugar group has been bonded to the nitrogen atom in an amino acid that is usually asparagine. Starts in the ER, finishes in the Golgi.

What is cotranslational folding? What are possible issues that can arise during cotranslational folding?

One way proteins fold up into a tertiary structure is through cotranslational folding in which polypeptides spontaneously fold up correctly as they are coming out of the ribosome to generate a functional protein. Cotranslational protein occurs in an aqueous environment so if a polypeptide has large hydrophobic domains, those domains will spontaneously aggregate to exclude water which can lead to a misfolded protein product that is nonfunctional.

What holds a lipid bilayer together? How/why does the structure of a phospholipid drive it to form a bilayer?

Polar molecules cluster together. The polar clusters repel the nonpolar molecules/structures, pushing the nonpolar molecules/structures together. The hydrophobic effect causes lipids in aqueous solutions to form bilayers or micelles

What are the differences between primary, secondary, tertiary and quaternary levels of protein structure?

Primary protein structure: Sequence of amino acids in a polypeptide chain. Secondary protein structure: Hydrogen bonding of the peptide backbone causes the amino acids to fold into a repeating pattern known as alpha-helix or beta-pleated sheet. Tertiary protein structure: Three dimensional folding pattern of a protein due to side chain interactions. Quaternary protein structure: Protein consisting of more than one amino acid chain.

How/why does a proteasome degrade just ubiquitin-tagged proteins? (not all proteins)

Proteasome is a large complex made up of a core and two caps. The target protein is degraded in the core which is a hollow cylinder. The proteasome cap has a binding site for the polyubiquitin chain. Ubiquitins are released from the target protein. The cap uses ATP hydrolysis to unfold the protein, feed it into the core. Target protein is degraded by proteases in the core of the proteasome. Even very large proteins (i.e. cytoplasmic dynein) are normally recycled in this way.

How do cells recycle ER-resident proteins back to the ER (from the Golgi)? (vocab: KDEL tag, COPI coat)

Proteins that normally localize to the ER have a sequence called a KDEL tag. So if the ER protein goes missing and winds up in a golgi cisterna when it's not supposed to be there, a KDEL receptor in the cisterna will bind to the KDEL sequence and recruit CopI coat proteins to bud off the membrane and form a vesicle that will take the rogue protein with the KDEL back to the ER.

How does ER shape correspond to ER function?

RER tends to be sheet-like. SER tends to be tubular.

Rab proteins are also GTPases. How are these activated? What makes it specific? ( i.e. why don't you see active Rab1 on all membrane compartments of the cell?)

Rabs are bound to soluble carrier proteins until a Rab-GEF drives the Rab to pick up GTP. GTP binding makes the Rab protein expose an alpha helix. Rab embeds in the nearest lipid bilayer as an integral membrane protein. Each type of membrane has a particular subset of Rabs associated. ER-Golgi vesicles are tagged with Rab1. Golgi-ER vesicles are tagged with Rab-2

Why is Ran GTP-associated inside the nucleus vs. GDP-associated when outside the nucleus?

Ran is GDP-associated outside the nucleus because Ran-GTP is hydrolyzed in the cytoplasm, because that's where GAP is. Ran is GTP-associated inside the nucleus because Ran picks up new GTP in the nucleus, because that's where GEF is.

Can you compare/contrast Importin and Exportin? (and how does Ran affect each of those?)

Ran-GTP drives a conformational change in: Importin promoting the release of cargo. Exportin promoting the binding of cargo.

What is glycosylation?

Reaction in which carbohydrate (oligosaccharide) is bonded to another molecule (e.g. to another carbohydrate, or to a protein or lipid)

What determines whether a protein translated at the ER is ultimately: 1) released into the ER lumen vs. 2) becomes integrated into the ER bilayer?

Released into the ER lumen: 7-12 hydrophobic amino acids Integrated into the ER bilayer: 20 hydrophobic amino acids

Can you explain the steps that ensure a protein is translated in conjunction with the ER? (including names What is the SRP, the SRP receptor, the translocon, etc and how do they help?)

SRP binds to ER signal sequence and blocks translation. SRP binds to SRP receptors; ribosome docks on membranes. GTP binds to SRP and SRP receptor; pore opens as the polypeptide is inserted. GTP is hydrolyzed and SRP is released. Signal sequence is cleaved by signal peptidase as polypeptide elongates and translocates into ER lumen. Completed polypeptide is released into ER lumen, ribosome is released, and translocon pore closes.

What distinguishes a saturated fatty acid from an unsaturated one?

Saturated fatty acid: No double bonds in their hydrocarbon tails. Unsaturated fatty acid: One or more double bonds in their hydrocarbon tails

Compare and contrast the benefits and weaknesses of scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

Scanning electron microscopy: Benefits: Great depth of field due to the fact that the electron scattering depends on the angle of the surface relative to the beam. Weakness: Only surface features can be imaged and unfortunately the resolution attainable is not very high. Transmission electron microscopy: Benefits: It has much better resolution. Weakness: Harder to prepare samples. First you have to permeabilize and fix the sample, then embed the sample in a resin block, after section with a diamond knife, and lastly take pictures.

Draw conclusions from evaluating experimental results (e..g. Understand which experimental procedure and outcome from investigating familial hypercholesterolemia (FH) led to the discovery of receptor-mediated endocytosis)

Since LDL metabolism is under negative feedback control, scientists wondered if it was broken in patients with FH. They wondered if the LDL between a normal and FH patient was different, rendering the FH LDL unable to regulate cellular cholesterol production. They obtained LDL from the blood of an FH and normal patient and put it into a surrounding media of cells in a petri dish. They measured whether the LDL from two individuals had different regulatory effects on the production of cholesterol within the cell. It resulted that FH patients had normal LDL and were able to regulate the production of cholesterol within the cell. Next the scientists took cells from a 12 year old suffering from FH and a healthy patient, to grow them in a petri dish. They wanted to examine how the presence or absence of LDL in the media would affect the cells' production of cholesterol. When LDL was absent, FH and normal cells produced large amounts of cholesterol. But, when LDL was added back the normal cells stopped producing cholesterol, meanwhile the FH cells kept up cholesterol production. This experiment suggests that the cell itself was not able to respond to LDL levels outside the cell, breaking the communication of the inside and outside of the cell. This led to the important discovery of the LDL receptor on the cell surface. LDL outside the cell binds to the LDL receptor, it is internalized and broken down to release cholesterol which can then inhibit the cholesterol producing enzyme from making more cholesterol. The LDL receptor then returns to the surface, making one round trip every 20 minutes which is an example of receptor mediated endocytosis. This experiment led to the discovery that FH is due to the mutation of the LDL receptor rendering it dysfunctional. Without this receptor, LDL outside the cell can't signal to the machinery inside the cell and stop its activity.

What sort of content cannot pass through a solid bilayer?

Solid bilayer is highly impermeable to ions regardless of the size. Ex. Ions: H+, Na+, HCO3-, K+, Ca2+, Cl-, Mg2+

What are the 2 different models for how proteins released from the ER move through the Golgi?

Stationary cisternae model: Each compartment of the golgi apparatus is a stable structure with enzymes in each cisternae held in place while the molecules in transit move through the cisternae in sequence carried by transport vesicles. A retrograde flow of vesicles then retrieves escaped ER and Golgi proteins that are necessary for their normal function and returns them to the preceding compartments. Cisternal maturation model: Views the Golgi apparatus as a dynamic structure in which the cisternae themselves move. The vesicular tubular clusters that arrive from the ER fuse together to one another to become the cis golgi network and this network progressively matures to become a cisterna and then a medial cisterna and so on. New cisternae continually form and then migrate through the stack as they mature. Retrograde flow explains the characteristic distribution of golgi enzymes. Everything continuously moves forward but CopI coated vesicles continually collect the appropriate enzymes and carry them back to the earlier cisternae where they function. When a cisterna finally moves forward to become part of the trans golgi network various types of vesicles bud off until the network disappears and at the same time other transport vesicles are continually retrieving the membrane from post golgi compartments that they are delivered to and returning this membrane to the trans golgi network.

How does an NLS help proteins interact with Karyopherin proteins?

Targeting of a protein to the nucleus requires a signal as part of the protein. Then karyopherins help NLS-tagged proteins get through the nuclear pore.

What does the fluidity of the lipid bilayer depend on? Explain the reasoning behind each item (temperature, ratio of saturated to unsaturated fatty acid chains, etc).

Temperature: Low temperature: The fluidity is low because the phospholipids are in a crystallized form due to low energy. High temperature: The fluidity is high because the phospholipids are loosely distanced due to high energy. Cholesterol: Low temperature: Increases the distance between phospholipids which increases fluidity. High temperature: Pulls phospholipids closer together to be able to attach to cholesterol which will decrease the fluidity. Saturated vs. Unsaturated Fats: Saturated: Are able to stack perfectly because of the straight chain which causes the phospholipids to be tightly packed, decreasing the fluidity. Unsaturated: Are able to stack with a bend because of the double bond which causes the phospholipids to be loosely packed, increasing the fluidity.

What is the "Fluid mosaic model"?

The fluid mosaic model describes the cell membrane as a tapestry of several types of molecules (phospholipids, cholesterols, and proteins) that are constantly moving.

What sort of phospholipid movements give rise to the "Fluid" part in the "Fluid Mosaic Model" of plasma membranes?

The fluid mosaic model describes the cell membrane as a tapestry of several types of molecules (phospholipids, cholesterols, and proteins) that are constantly moving. This movement helps the cell membrane maintain its role as a barrier between the inside and outside of the cell environments.

Name the parts the Golgi (Which part is closest to the ER? Etc.)

The part facing towards the ER is called the cis Golgi network. cis Golgi medial Golgi trans Golgi The part facing towards the plasma membrane is called the trans Golgi network.

Is the ubiquitin pathway only used for protein degradation?

The ubiquitin pathway is not only used for protein degradation. It can be used to signal a protein that needs to be repaired or endocytosed.

Can you comfortably design an indirect immunofluorescence experiment?

This can be used to strategically highlight proteins (with primary antibodies). Fluorophores are then attached to the primary antibodies (with secondary antibodies). Making a primary antibody: 1. Inject specific "antigen" into rabbit 2. Extract antibodies from rabbit Making a secondary antibody: 1. Inject lots of rabbit protein (antigens) into goat 2. Extract antibodies from goat 3. Attach fluorophores to the antibodies

What makes it possible for alpha helices and beta barrels to embed in a bilayer? How is the polar character of the protein backbone masked by these structures? What makes the surface of these proteins compatible with bilayer interior?

Unfolded the protein has peptide bonds that are polar. Once folded the alpha helix hides the polar backbone of the polypeptide chain. Then the R groups stick out, being non-polar they are compatible with the hydrophobic interior of the bilayer.

Why is the emission light of a fluorophore always a longer wavelength/lower energy than excitation light?

When electrons go from the excited state to the ground state there is a loss of vibrational energy. As a result, the emission spectrum is shifted to longer wavelengths than the excitation spectrum.

How does bacteria accomplish homeoviscious adaptation?

When temperatures are cold bacteria activate enzymes that remove two carbons from an 18 carbon fatty acid chain to turn into an 16 carbon fatty acid chain. Shorter fatty acid chain is associated with increased membrane fluidity. When temperatures are cold e. coli activates desaturase enzyme which removes hydrogens from and creates double bonds between carbons of fatty acids.

How do reptiles accomplish homeoviscious adaptation?

When temperatures are hot, reptiles increase the proportion of cholesterol and unsaturated lipids in the cell membranes.

Receptor-mediated endocytosis

also called clathrin-mediated endocytosis, is a process by which cells absorb metabolites, hormones, proteins - and in some cases viruses - by the inward budding of the plasma membrane (invagination).

What is a uniporter?

move one type of solute across the membrane, down its concentration gradient (therefore it is not a pump).

What is antiporter?

move solutes in the opposite direction across the membrane (pump).

What is symporter?

move solutes in the same direction across the membrane (pump).

Phagocytosis

the ingestion of bacteria or other material by phagocytes and amoeboid protozoans.

Pinocytosis

the ingestion of liquid into a cell by the budding of small vesicles from the cell membrane.