Jan19- on (work in progress) MDSC 351 Content

Exam 1 content MDSC 351

Describe the nuclear envelope

It separates the genetic material from the cytoplasm, has two membranes that are fused at NPC, the outer membrane is studded with ribosomes, and is continuous with the rough ER. Filaments of the cytoskeleton are attached to the outer membrane.

What is the role of fascin or F-actin?

A binding protein that regulates the adhesion of migrating cells. It binds directly to nesprin-2 at the nuclear envelope and together they regulate nuclear moving in migration. If you lose fascin-nesprin complex, the nucleus deforms and cell invasion is reduced (migration of cells within a tissue).

What are lamins?

A special class of intermediate filaments that polymerize into a 2D lattice

What is the nuclear lamina?

A meshwork of interconnected nuclear lamins that is anchored to the inner membrane of nuclear envelope. Main function: it gives shape and mechanical stability and disassembles prior to mitosis.

What are is the nuclear lamina regulated by?

Phosphorylation

What happens if cells have truncated/mutant lamin?

Misshapen nuclei

What was the evidence for movement of material past the nuclear envelope?

Gold particles were coated with a protein normally found in the nucleus. These were injected into a frog cell and the particles were observed gong through nuclear pores

What kinds of molecules move across the nuclear envelope?

Proteins for replication and transcription (made in the cytoplasm, exported to the nucleus). RNA molecules move through the NPC, mRNAs, tRNAs, rRNAs are made in the nucleus and must be transported to cytoplasm. Spliceosome components have to go both ways.

Describe the structure of the nuclear pore complex

It projects into the cytoplasm and nucleus, and is a large, complex structure made up of about 30 different proteins (nucleoporins). FG nucleoporins line the channel and block diffusion of large molecules. A transmembrane ring holds the NPC to the nuclear envelope.

Elaborate on the process that allows for unidirectional transport of RNA transcripts out of the nucleus

There are RNA processing platforms: one basket platform on the nucleus side that enables RNA processing and export. The second is on the cytoplasmic that has other RNA remodeling proteins and allows for RNA export.

What are nuclear localization signals?

Classical NLSs have 1 or 2 short stretches of positive amino acids. Anything lacking one of these will not be localized to the nucleus.

Describe transport through the nuclear pore complex of a protein with a nuclear localization signal

In the cytoplasm a protein with NLS binds to an alpha/beta importin dimer and their complex docks with the NPC filaments that extend into the cytoplasm, and then the complex moves through the nuclear pore by interacting with the FG domains of FG nucleoporins.

What is the selectivity of the NPC like?

Rapid passage for anything bound to nuclear transport receptors, but inert macromolecules greater than 40kDa cannot pass.

How could NPC selectivity be explained by its structure?

There was a proposal of a sieve-like structure created via cross linking between FG rich nucleoporin repeats and FG domains would provide binding sites for the facilitated diffusion of nuclear transport factors with molecular cargos.

After the molecular cargo-importin complex passes through the NPC, what happens in the nuclear compartment?

The complex interacts with Ran-GTP which causes its disassembly (yay delivery complete!). The importin beta associated with Ran-GTP goes back to the cytoplasm where the Ran-GTP is hydrolyzed to Ran-GDP. Ran-GDP is transported back to the nucleus and importin alpha associates with an exporting and returns to the cytoplasm.

What is Ran? What are its two forms?

GTP binding protein that exists in two forms. The two forms are Ran-GTP (active) and Ran-GDP (inactive).

What is a GEF protein?

Guanine nucleotide-exchange factors (GEFs). These bind to inactive G protein and stimulate the dissociation of the GDP bound to it. Once the GDP is released, the G protein can bind a GTP.

How does the steep Ran gradient (lots of Ran-GTP in nucleus, low concentration in cytoplasm) drive nucleocytoplasmic transport?

The energy released by GTP hydrolysis is used to maintain the Ran-GTP gradient. This gradient drives nuclear transport by a process that depends only on receptor-mediated diffusion; no motor proteins or ATPases have been implicated

What do the two different forms of Ran do for nucleocytoplasmic transportation?

They confer directionality to transport by dictating where importins and exporting bind and release cargo.

Why does the cell maintain a high concentration of Ran-GTP in the nucleus and a low concentration in the cytoplasm?

Due to compartmentalization of accessory proteins GAP and GEF. RanGAP1 is in the cytoplasm, where it promotes the hydrolysis of Ran-GTP to Ran-GDP, thus maintaining the low cytoplasmic level of Ran-GTP. RanGEF is found in the nucleus, and it converts Ran-GDP to Ran-GTP, thus maintaining the high nuclear level of Ran-GTP.

Where is there high concentration of Ran-GDP?

Cytoplasm

What are nuclear export signals?

Are leucine rich (much less studied than NLS). Proteins exported from the nucleus contain these signals.

You take the normal sequence of amino acids that make up the NES and fuse it to a nuclear protein. You then inject this new fused protein into the nucleus. What happens to this protein?

It moves into the cytoplasm.

What is the nuclear matrix?

More than a skeleton, it maintains the shape of the nucleus and organizes the chromatin. It also anchors machinery for replication, transcription, RNA processing. Allows chromatin to acquire or maintain the open conformation necessary for transcription.

What happens in the nucleolus?

Site of rRNA synthesis - ribosomal subunits in various stages of assembly give the nucleolus its granular appearance

Explain how chromatin is packaged in the nucleus

In the nucleosome - which is supercoiled DNA wrapped twice around histone complexes and adjacent nucleosomes are connected by linker histones and linker DNA.

What are histones?

They are conserved positively charged proteins that interact with the negatively charged DNA backbone. They improve DNA stability and how tightly wound DNA is around them impacts how open it is to transcription. In addition, DNA coils around them more tightly during mitosis.

What can DNA loops bring together?

They can bring together enhancers and promoters, and loci of genes.

True or false: the nucleus is a randomly positioned sack of components

Chromosomes occupy distinct territories, and different

Explain the functions of the plasma membrane

Compartmentalization, serving as a scaffold for biochemical activities, selectively permeable barrier, transporting solutes, responding to external signals and intercellular interaction.

What is the fluid mosaic model?

A lipid bilayer with the H2O soluble heads facing outwards and the hydrophobic tails facing in. The membrane proteins occur as a mosaic discontinuous particles that penetrate the sheet, cellular membranes are dynamic structures where both lipids and proteins are mobile and can interact with other membrane molecules.

What determines the protein to lipid ratio of the plasma membrane?

Depends on membrane type, organism and cell. Neurons have a low protein to lipid ratio to insulate electric signals while mitochondrial inner membranes have high protein to lipid ratios.

What is the plasma membrane composed of?

Lipid bilayer is the structural backbone of the PM, proteins carry out most of the PM’s specific functions, and there are carbohydrates

Describe phosphoglycerides

Built on a glycerol backbone. Diglyceride with two

What are the three types of membrane lipids?

Phosphoglycerides, sphingolipids, and cholestrol

Describe the types and roles of plasma membrane lipids, carbohydrates, proteins

The membrane lipids - membrane fluidity, membrane composition, compose the lipid bilayers

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The membrane carbohydrates - Bound to membrane lipids or proteins, assist in identification, specificity of reactions.

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The membrane proteins - (integral, peripheral, lipid anchored) can serve structural roles, serve as receptors, transporters, enzymes and cell signalling.

What is the transition temperature?

The temperature at which the membrane will change from a fluid liquid-crystal state to a frozen crystalline gel.

What can increase membrane fluidity?

If you have shorter fatty acid chains = you have a lower transition temperature.

Desaturation, i.e., presence of double bonds = lower transition temp, the lower the temperature at which the bilayer gels

LASTLY, Cholesterol tends to abolish sharp transitions and promotes an intermediate fluidity

Importance of membrane fluidity?

The physical state of the PM affect function: compromise b/w a rigid ordered structure (without movement) and a

completely non- viscous liquid (with no organization or

mechanical support).

Membrane fluidity allows interactions to take place in the membrane (clustering and movement).

Membrane fluidity plays a key role in membrane assembly & repair: Needed in many basic processes (movement, growth, division, secretion, endocytosis)

Elaborate on why membrane domains matter

Different PM regions carry out different functions.

Example: an epithelial cell in the intestine is a
polarized cell: apical, basal and lateral domains

What general principles govern how solutes can be moved across the membrane?

Depends on presence of a concentration gradient and the ability of the solute to be able to cross the phospholipid bilayer. Movement of charged species additionally depends upon concentration and electrical gradients.

Why does the movement of charged species across membranes depend on concentration and electrical gradients?

Ions will repel like charges, and get attracted by opposite ones, so the electrochemical gradient matters.

What are the differences between ion channels and transporters?

1. Ion channels do not use ATP, some types of transporters do.
2. Transporters involve conformational changes in the transporter protein itself due to ion binding,
3. Active trasnporters also couple conformational changes to physical movement pf the ions across the membrane bilayer.

An important aspect of transport by facilitated transporters and pumps is ______

Conformational shifts

What is cotransport aka secondary transport?

The coupling of active transport to existing concentration gradients. The existing concentration gradients of ions can be used for their potential energy to transport other solutes.

Describe the Na+ coupled glucose transporters

Glucose is transported and concentrated from the lumen of the intestine to the cytoplasm (and then into the blood stream). This mechanism depends on the maintenance of the Na+ gradient by the Na+/K+ ATPase pump .

In a Na+/Glucose cotransporter, the ____ moves down its concentration gradient, driving the transport of the ____ against its gradient

Na+, Glucose

What are symports?

Move solutes the same direction

What are antiports?

Moves solutes in opposite direction

What are the similarities between ion channels and transporters?

1. Facilitated transporters and ion channels do not use ATP
2. Work based on concentration gradient
3. Faster than simple diffusion

What are the types of transporters?

Facilitative, active, secondary active

What does a facilitative transporter do?

Moves solutes from one side to the other, at a rate faster than simple diffusion via substance-specific membrane protein. This is specific and passive.

How does the Na+/K+ ATPase pump work?

For each ATP hydrolyzed: 3 Na+ OUT and 2 K+ IN. This occurs by Na+ binding INSIDE the cell and indicted changes via ATP hydrolysis gets it released OUT. Then the K+ OUTSIDE can bind to the changed pump, ATP binding means that the K+ is released INTO the cell.

What happens in the Na+/K+ ATPase pump when the pump in E1 gets occluded?

Na+ cannot flow back into cytosol, leads to ATP hydrolysis and the phosphorylation of protein and ADP release makes E1 change to E2 conformation.

What happens in Na+/K+ ATPase pump after E1 has changed to E2?

The binding sites are now open to the extracellular environment, and E2 pumps are less attached to Na+, releasing those ions. E2 instead picks up 2 K+.

What happens in the Na+/K+ ATPase pump when E2 gets occluded?

Another change in conformation, prevents K+ flow back into the extracellular space. Dephosphorylation happens followed by ATP binding that gets the pump to change back to E1.

What is the difference between E1 and E2 conformations on the Na+/K+ ATPase pump?

E1 conformation has ion binding sites open to the inside of the cell, and the E2 conformation has ion binding sites open to the outside.

Where is K+ concentrated in the cell?

Inside the cell

Where is Na+ concentrated in the cell?

Outside the cell

What kind of pump is the Na+/K+ pump?

Acceptable answers: Active transporter, but also a P-type pump (phophorylation causes changes in conformation and ion affinity).

For the H+/k+ ATPase pump in the cells lining the stomach, what does food stimulate?

Food simulates the release of histamine, which causes the pumps to be fused to the PM, activating the H+/K+ pump that pumps H+ into the stomach against the concentration gradient.

What is an active transporter?

Transports a specific solute against its concentration gradient by using the energy of ATP hydrolysis. These are integral membrane proteins selectively bind in a particular solute and moves it across the PM in a process driven by changes in confirmation. Allows passage against a concentration gradient.

How does facilitated diffusion work with a glucose transporter?

The glucose transporter in the outward-facing conformation binds glucose changes to the inward-facing conformation and releases its substrate inside the cell. Empty transporter shuttles between inward/outward conformations

What is a secondary active transporter?

Couples transport with the energy of an electrochemical gradient.

What is facilitated diffusion?

A type of diffusion that is faster than simple diffusion using a substance-specific membrane protein (aka facilitative transporter). This is specific AND passive. Depends on concentration gradient

Describe the functions of ion channels

These are transmembrane structures permeable to specific ions (especially necessary ones that cannot cross the bilayer) e.g. K+. Ion channels can be highly specific, and some are called gated because they open in response to various stimuli.

What are the types of ion channels?

Gated channels:

* voltage gated- depends on ionic charge difference across PM


* ligand gated- depends on the binding of a specific molecule (not the solute passing through)
* Mechano gated - depends on mechanical forces like stretch
* Gap junctions - allows various molecules to directly pass through a regulated gate between cells.

Do ion channels require input of energy?

No.

How does the structure of the K+ ion channel inform function?

Selects for K+ versus Na+ ions, the oxygen atoms of he carbonyl groups of the subunits interact selectively with K+.

What would be the differences for proteins/parts of the bilayer inside the membrane versus outside?

Lipids lacking polar groups can move across the membrane, glycosylated or disulfide bonded residues will never be found on the cytosolic side of the protein, glycosylation or disulfide bonds only found on proteins OUTSIDE the membrane.

Why are oligosaccharides on the surface of the plasma membrane can be involved in specific interactions, polysaccharides such as starch and glycogen, are not?

Oligosaccharides are heteropolymers, have longer lengths and different connectivity. Glucose and starch = monopolymers.

What are the two main types of membrane transport?

Passive and active transport.

What decides how certain \n molecules are being \n transported?

Small, inorganic molecules pass readily across membranes by slipping between phospholipids. E.g. O2 , CO2 , H2O, NO) \n

Small molecules such as sugars and amino \n acids diffuse very slowly across membranes. Polarity is an important factor! They might need to be transported. \n

What is the partition coefficient and what does it mean for membrane transport?

The partition coefficient = solubility in a nonpolar \n solvent : solubility in water. Permeability is \n generally proportional to lipid solubility

If a substance partitions preferentially into \n octanol over water, what is likely to be its membrane permeability?

It will have higher membrane permeability because it is more hydrophobic

What are the ways to move substances across membranes?

Simple diffusion, facilitated diffusion, active transport

What is simple diffusion?

Spontaneous processes by which a substance moves down its concentration gradient. Only happens for small, uncharged molecules (less polar move faster), and the membrane must be permeable to the substance.

What are aquaporins?

Cells move water faster than what diffusion would accomplish, these are channels that have positive charges inside them (serves a sieve, preventing anything that isn’t water from going through).

What is the endomembrane system?

The membrane system within the cell, where the \n individual components function together as a \n coordinated unit.

List the components of the endomembrane system?

Endoplasmic reticulum. \n • Golgi complex. \n • Vesicles. \n • Lysosomes. \n • Endosomes. \n Not mitochondria!

What are the two parts of the endocytosis/secretory pathway through the cytoplasm?

Constitutive and regulated secretion

What is constitutive secretion?

Forms the extracellular matrix \n • Formation of the PM or \n organelle membranes.

What is regulated secretion?

Material stored in membrane- \n bound packages and secreted \n in response to a particular \n stimulus.

What is the endocytic pathway?

Movement of material from outside, into the cell \n and into organelles such as endosomes and \n lysosomes.

What is the endoplasmic reticulum?

Responsible for protein and lipid biosynthesis. Connected with the outer nuclear membrane. There are two types (smooth and rough).

What is the rough ER/what does it do?

It is continuous with the outer membrane of the nuclear envelope, with ribosomes on its cytosolic surface. Made up of a network of flattened discs called cisternae. Abundant in cells with secretory functions such as mucous-secreting cells lining the digestive tract.

What is the smooth ER/what does it do?

Tubular structure that forms a network of “pipelines” through the cell. \n Functions: \n • Steroid hormone synthesis in the gonads or \n adrenal cortex. \n • Detoxification of compounds in the liver. \n • Sequesters Ca2+ in muscle cells, regulated release causes muscle contraction.

What proteins are made by the Rough ER?

– Integral membrane proteins. \n – Soluble proteins to be secreted. \n – Soluble proteins that will reside within organelles

Which proteins are made by free ribosomes in the cytoplasm?

– Enzymes, cytoskeletal proteins, peripheral proteins of \n the inner PM, proteins destined for nucleus, proteins \n to be incorporated into mitochondria and peroxisomes

How does the cell know where to make certain proteins?

Secretory proteins have built-in \n address codes. A sequence of amino acids at their N-terminus that direct the emerging polypeptide and ribosome to the ER membrane. The polypeptide moves into the cisternal space of the ER through a channel in the ER \n membrane

Describe the process of targeting and synthesizing proteins in the RER

The first step is synthesis beginning on a free ribosome. As the signal sequence emerges, a sequence recognition particle (SRP) recognizes it. SRP binding temporarily arrests further translation. The SRP-ribosome complex binds with an SRP receptor on the ER membrane. The ribosome then binds with the translocon on the ER, releasing the SRP.

The signal peptide binds to the inside of the translocon, \n The signal peptide binds to the interior of the \n translocon and contact between the translocon and polypeptide allows the growing polypeptide to enter the ER lumen, where the signal peptide is cleaved and the protein is folded with the help of chaperones.

What is the effect of GDP-binding versus GTP-bound proteins on synthesis and trafficking of secretory proteins?

GTP-bound and GDP-bound G protein \n have different conformations. \n The GTP-bound protein typically turns \n the process on, and hydrolysis of the \n bound GTP turns it off. \n SRP and the SRP receptor are G \n proteins that interact with one another \n in their GTP-bound states; GTP \n hydrolysis triggers the release of the \n signal sequence by the SRP.

What is the translocon?

A complex of proteins, this most commonly refers to the complex that transports nascent polypeptides with a targeting signal sequence into the interior space of the endoplasmic reticulum from the cytosol.

How are integral membrane proteins synthesized?

Integral membrane proteins are synthesized co- \n translationally. Their hydrophobic transmembrane are shunted from the translocon to the lipid bilayer.

During synthesis, the inner lining of the translocon orients the nascent polypeptide so the positive end faces cytosol. \n In multispanning proteins, sequential transmembrane \n segments typically have opposite orientations, so \n their arrangement in the membrane is determined by \n the direction in which the first segment is inserted.

Tail-anchored proteins lack a signal sequence, but \n are synthesized in the cytoplasm, and targeted to the \n ER through interactions with proteins in the GET pathway

What does it mean that proteins are synthesized cotranslationally?

Translocation, modification occuring while the protein is being translated.

Where does mannose-phosphorylation happen?

In the Golgi.

How are new proteins processed in the ER?

The signal peptide is cleaved off by signal \n peptidase.

Carbohydrates are added: This is important to protein function (binding sites), and aids in proper folding. \n \n Molecular chaperones in ER lumen: recognize and \n bind to unfolded or misfolded proteins and help \n these to fold properly

\n __There are protein-processing enzymes__ in the ER. \n Example: proteins enter the lumen with cysteine in \n the –SH state but leave the ER with many of the \n residues joined as disulfides (-SS-).

Why are the –SS- residues of PM integral proteins always \n extracellular?

Describe membrane biosynthesis

Newly synthesized proteins and lipids are inserted \n into existing membranes of the ER \n • Membranes can be enzymatically modified as they \n move from ER into other cellular compartments \n • Most lipids are made within the ER \n Sphingomyelin and glycolipids: synthesis begins in \n ER, finished in Golgi \n Unique mitochondrial lipids: made on mitochondrial \n membrane

How does retrieval of escaped ER \n proteins via COPI vesicles?

Soluble ER proteins, such as \n BiP, bear an ER retrieval \n sequence on their c-terminus, \n typically Lys-Asp-Glu-Leu \n (KDEL). \n • Escaped ER proteins are \n recognized by “KDEL \n receptors”, in the Golgi which \n recruit COPI coat proteins and \n result in return of these \n proteins to the ER.

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