cell bio exam 3: membranes + transport

What is lateral diffusion?

rapid, horizontal movement of lipids and proteins within a leaflet/monolayer

What factors regulate membrane bilayer fluidity?

• chain length:

  • longer chain = more rigid

  • shorter chian = more fluid

• saturated vs. unsaturated:

  • saturated = straight, packed, more rigid

  • unsaturated = kink/bend, more fluid

• temperature:

  • cold = more rigid

  • warm = more fluid

• cholesterol

  • too rigid → breaks up tight/packed interactions → introduces fluidity

  • too fluid → restricts movement of fatty acids —> introduces rigidity/integrity

How do phospholipids move

• lateral: flexion, rotation 

• transverse: flip-flop (rarely occurs)

what are the three categories of membrane proteins? how do they interact with membrane bilayer?

• integral: spans entire membrane bilayer; strong hydrophobic interactions with hydrophic tails; often alpha helical or beta barrel structures

• peripheral: loosely attached to membrane surface via. hydrophilic interactions w/ hydrophilic polar heads

• lipid-anchored: post-translational modification → covalently attached lipid that anchors protein onto membrane

How is membrane fluidity measured?

FRAP: fluorescence recovery after photobleaching

1. outer leaflet is fluorescently labeled

2. bleach area with laser

3. monitor recovery of fluorescence in the bleached area

results

• fast recovery: high lateral mobility and membrane fluidity

• slow/no recovery: restricted mobility, possibly mobility

What mechanisms restrict protein localization within membranes?

• tethered to cell cortex — proteins anchored to cytoskeleton

• tethered to extracellular matrix

• tethered to proteins on adjacent cell matrix (cell-cell adhesion)

• diffusion barriers (tight junctions) — restrict lateral diffusion of proteins and lipids; segregates between apical and basolateral membrane domains

What is glycocalyx?

• sugar coating that reinforces plasma membrane

• always on extracellular leaflet on plasma membrane?

What are glycoproteins?

• transmembrane protein w/ short, branched chain of sugars covalently attached to surface of plasma membrane

• function: communication and recognition

What are proteoglycans?

• transmembrane protein with long, unbranched polysaccharide chain

• function: structural and signaling functions in the extracellular matrix

What is cell cortex?

• framework of cytoskeletal proteins attached to plasma membrane

• function: structural support, cell shape, anchors transmembrane proteins tethered to cortex,

What are tight junctions?

• formed by claudin protein

• forms a barrier between adjacent cells to control the movement of

• prevents movement of proteins and lipids between apical and basolateral sides to maintain cell polarity

• found in epithelial and endothelial tissues

what is apical-basal polarity?

apical: faces external environment (gut cavity)

basolateral: faces interior environment and bloodstream

what is passive transport?

• transport with concentration gradient (high to low conc)

• no energy required

what is simple diffusion?

• passive transport

• small, uncharged (nonpolar + polar) molecules pass freely through plasma membrane

• e.g., O₂, CO₂, H₂O

• no transporter required

what is facilitated diffusion?

• passive transport through channels or transporters

• no energy required

what is channel-mediated transport?

• transport of selective ions down concentration gradient (passive transport)

• channel either open or closed conformation

• fast movement of LOTS of ions

what is transporter-mediated transport?

• SLOW transport — moves one or few molecules at a time

• when open to outside, closed to inside

• passive transport

what is active transport?

• movement of molecules against concentration gradient (low to high conc.)

• requires energy

• uses pumps to transport ions/molecules

What are different types of active transporters?

• symporter: both molecules move same direction and against their conc. gradient

• antiporter: molecules move in diff directions and against their conc. gradient

  • switches between two conformational states ONLY if one binding site is occupied

  • does not switch conformational states if BOTH/NO binding sites are occupied

**both are secondary active transport

what is the sodium-potassium pump?

• use ATP to move Na⁺ and K⁺ against their concentration gradient

• 3 Na⁺ out, 2 K⁺ in

what is sodium-proton exchanger (NHE)?

• exchanges one intracellular H⁺ (proton) for one extracellular Na⁺.

• antiporter

• maintains neutral intracellular pH by removing excess H⁺ in cytosol

What is GLUT?

• uniporter that facilitates diffusion of glucose down its concentration gradient

• located at basolateral membrane of intestinal epithelial

What is SGLT?

• sodium-glucose symporter

• couples glucose uptake against its gradient with Na⁺ moving down its conc. gradient

• located at apical membrane

• glucose and sodium move from intestinal lumen to epithelial cell

what are the different ways that ion channels can be gated?

ligand-gated: molecule binds to channel and induces conformational charge

mechanically-gated: protein-protein interaction forces channel open

voltage-gated: channel opens when there is a change in membrane potential

what is membrane potential?

difference in electrical charge between the surface of the outside and inside of the membrane

what is patch clamping?

• measures movement of ions through ion channel (channel activity

• change in current when channel is open

what is signal propagation?

1. opening of one ion channel increases positive charge of membrane potential

2. relays signal and stimulates opening of neighboring channel

how is an action potential triggered?

1. rapid depolarizing stimulus triggers change from -60 mV to -40mV (threshold potential)

2. when membrane reaches threshold potential, voltage-gated Na⁺ channel opens → Na⁺ rushed into cell and increases membrane potential

3. @ +40 mV repolarization occurs

• voltage-gated Na⁺ channels inactivate → no more Na⁺ enters

• voltage-gated K⁺ channels open → K⁺ leaves cell and membrane potential is lowered towards resting membrane potential

4. hyperpolarization

• Na⁺ channels are closed but not locked

• K⁺ channels are slow to close → extra K⁺ leaves

• membrane potential briefly becomes more negative than resting potential (-80 mV)

• Na⁺/K⁺ pump and leak channels gradually restore the resting potential

5. refractory period: no action potential will be stimulated during this time

what are the conditions of a cell at resting state?

• resting membrane potential: -70 mV

• Na⁺/K⁺ and leak channels maintain high K⁺ inside and high Na⁺ outside

• voltage-gated Na⁺ and K⁺ channels are closed

how are neurotransmitters released?

1. action potential reaches presynaptic terminal and depolarization opens voltage-gated Ca²⁺ channels

2. Ca²⁺ rushes into presynaptic terminal → triggers neurotransmitter release

3. neurotransmitter-storing vesicles are docked at membrane; SNARE protein help position vesicle

4. vesicle membrane fuses with presynaptic membrane; neurotransmitters are released into synaptic cleft via. exocytosis

5. neurotransmitters diffuse across synaptic cleft and bind to receptors on postsynaptic membrane

What is Co-IP?

• co-immunoprecipitation: observes protein interactions

1. cell lysis to release proteins w/o disrupting interactions

2. antibody bindsb to target protein (“bait”)

3. precipitation: antibody-protein complex pulled out of solution

4. wash to remove unbound protein

5. elute + detect what other proteins bound to “bait” were pulled out of solution

what is co-translational synthesis?

translation/protein synthesis begins in the cytosol, resumes in endoplasmic reticulum, and then processed + secreted

how is co-translational synthesis initiated?

ribosome translates mRNA in cytosol

1. SRP (signal recognition particle) binds to ribosome and signal sequence as it emerges from ribosome (typically hydrophobic) → pauses translation temporarily

2. SRP-ribosome complex binds to SRP receptor on ER membrane

3. SRP is released → peptide passed to protein translocator and protein is threaded across ER membrane via. channel in translocator

4. signal sequence stays in translocator, exits lateral gate, and cleaved by transmembrane signal peptidase

5. translation resumes and growing polypeptide is threaded into ER lumen or inserted into ER membrane

What is soluble ER protein? What is their fate?

soluble ER protein: protein that is freely floating in ER lumen

fate -

1. undergo folding with chaperones and post-translational modifications

2. may either stay in ER, packaged into vesicles + transported to organelles, or secreted from cell

what is a single-pass transmembrane protein?

• goes through membrane ONCE

• has ONE transmembrane domain

what is the orientation of a single-pass transmembrane protein w/ cleavable signal sequence?

N-terminus in lumen of ER

C-terminus faces cytosol

what is the fate of single pass transmembrane proteins with cleavable signal sequence?

1. as polypeptide is threaded into ER lumen, hydrophobic stop-transfer sequence enters translocator → stops further translocation of segment into ER lumen

2. hydrophobic segments laterally exits translocator

3. hydrophobic sequence pushes ribosome off ER membrane → translation resumes in cytoplasm ribosome

what happens to single pass transmembrane protein w/o signal sequence?

• SRP recognizes transmembrane domain (hydrophobic segment) when it emerges from ribosome and inserts protein into membrane

• hydrophobic region fully spans membrane

• orientation depends on location of (+) charged amino acids

  • terminus with more (+) charged amino acids faces cytosol

  • bc electrostatic interactions with negatively charged cytosol

• ribosome continues translating remaining portion of protein

how are multipass transmembrane proteins inserted?

• FIRST signal sequence/transmembrane domain dictates orientation

• subsequent transmembrane domains follow in/out protein

what is glycosylation?

• post-translational modification where glucose is covalently attached

• nonspecific glycosylation occurs in ER

functions

• chaperones bind to specific sugar groups to help proteins fold properly

• glycosylation in cytoplasm indicate misfolding → triggers ERAD

what happens when there are misfolded proteins (small-scale)?

*glycosylation in cytoplasm is a marker for misfolded protein → triggers ERAD (ER-associated degradation)

1. glycanase cleaves glycosyl group from protein

2. ubiquitinase post-translationally modifies protein by covalently attaching ubiquitin (marker for protein degradation)

3. proteasome degrades protein and recycles amino acids

what happens when there are misfolded proteins (large-scale)?

*misfolded protein binds to receptor on ER membrane → activates UPR (unfolded protein synthesis)

1. reduce protein synthesis → less new protein enter ER

2. increases production of chaperones and ERAD machinery → help refold/remove misfolded protein

3. trigger apoptosis if stress can’t be reused
   

how does vesicular transport work?

1. vesicle buds off from donor compartment (e.g. ER, golgi)

2. vesicle moves through cytoplasm

3. vesicle fuses with target compartment and delivers cargo (e..g proteins, lipids)

what are coat proteins?

• proteins that bind to transmembrane protein to bend ER membrane into curve

• accumulation of coat protein causes vesicle budding

what is the difference between the two types of coat proteins?

COPII: moves vesicle from ER → golgi

COPI: moves vesicle from golgi → ER (retrograde transport) 

why is COPII nonspecific?

ALL proteins found in ER are transported to golgi for processing

what happens if COPII is inhibited?

protein is stuck in ER

what are examples of proteins that would be retrogradely transported?

• chaperones

• signal receptor particle (SRP receptor)

• translocator

• signal peptidase

What is KDEL?

• ER retrieval/retention signal that signals proteins are destined to stay in ER

• short amino acid sequece (Lys-Asp-Glu-Leu) at the C-terminus

• KDEL binds to KDEL receptor on golgi to induce conformational change that allows COPI to bind; accumulation of COPI induces membrane bending and vesicle budding

what happens if KDEL is inhibited?

KDEL ER protein is stuck in golgi

plasma membrane protein is secreted with no problem

How does fusing work?

*after budding, uncoating (removal of coat protein from vesicle) occurs, exposing surface proteins (e.g. v-SNAres, Rab-GTPases)

1. Rab GTPase binds to tethering proteins on target membrane → pulls vesicle towards target

• every target membrane has different tether proteins

• every rab binds to specific tether

2. docking — v-SNARE (vesicle) binds to t-SNARE (target) → zipping pulls membrane extremely close together

3. hemifusion — outer leaflets merger, inner leafletes remain separate

4. full fusion — inner leaflets fuse to form fusion pore → vesicle content is delivered to target and vesicle membrane is incorporated into target membrane

5. SNARE complex is disassembled by NSF and SNAP

How does patch clamp works?

use fine pipette to isolate a part of membrane and measure change in action potentials within the region

What is CTFR mutation? How can a drug resolve this?

• mutation causes protein misfolding and prevents from reaching cell surface

• Cl^- channel is defective → Cl^- can’t enter the cell

• results in thick mucus

resolution

• help CFTR deltaF508 fold

• increase UPR chaperone expression → helps with folding correctly

• increase expression of other Cl^- channels

How does cholesterol stiffen the membrane?

forms stable hydrophobic interactions between rigid fused ring structure of cholesterol and hydrophobic acyl tails of phospholipids

what mutations could explain accumulation of protein in vesicles?

• mutation on Rab

• mutation on v-SNARE

what mutations could explain accumulation of protein in golgi?

• mutation of COPI

• mutation of ER retention signal on C-terminus

what does oubain do?