BIO130 second half

Lec 13: membrane structure: animal vs plant cell specialties

Animal cell:

• Extracellular matrix

• Lysosomes — degradation of cellular components

.

Plant cell:

• Cell wall — shape and anti-stress

• Vacuoles — degradation (like lysosome) and storage

• Chloroplast — photosynthesis

Cytoplasm, Cytosol, and Lumen

Cytoplasm

• contents of the cell outside the nucleus (cytosol + organelles)

.

Cytosol

• Aqueous part of the cytoplasm (no organelles)

.

Lumen

• Inside of organelles

Cellular functions at membranes

1. Divide cell into compartments

2. Scaffold for biochemical activities (e.g. mitochondria)

3. Selectively permeable barrier + transport proteins regulate transport

4. Receptors respond to external signals

5. Proteins for interactions between cells

Membrane bilayers

Bilayer

• composed of amphipathic molecules (hydrophilic and hydrophobic parts)

  • hydrophilic head

  • hydrophobic tail

• spontaneously assemble into bilayer

  • hydrophilic head interacts with aqueous outside

  • hydrophobic tails interact with eachother on inside

  
  

• proteins and other stuff inside —→ fluid mosaic model

Types of membrane lipids

Phospholipids, sterols, glycolipids

• Different groups on the molecule

• All are amphipathic

Phospholipids

Many types of phospholipids

Hydrophilic head:

1. Different groups

2. Phosphate

3. Glycerol

Hydrocarbon tail

• length: 14-24

• saturated/unsaturated

• cis double bond leads to kink

Sealed compartments of bilayers & Liposomes

After assembly into bilayer, form into a sphere

Liposomes: artificial lipid bilayers

• Used for

  • studying lipid movement

  • protein properties

  • drug delivery into cells

Cell membranes are fluid

Can be deformed without damage

Phospholipid movement

(leaflet: one side of bilayer)

Phospholipids in one leaflet can

• move laterally

• rotate

• flex

Rarely..

• move from one leaflet to another (flip flop) on their own

  • hydrophilic head has to move through hydrophobic core

  • not energetically favourable

.

Membrane fluidity regulated to keep it consistent

Membrane has many proteins

Factors affecting membrane fluidity

Temperature

• lower temp — less fluid

• more important for single-celled

Composition

• phospholipid saturation — unsaturated more fluid (kinks give space)

• tail length — some shorter more fluid

• lipid composition — cholesterol stiffens, less permeable to water

Sterols — Cholesterol

Animals — mainly cholesterol

Plants — plant sterols, some cholesterol

.

There can be up to 1:1 ratio of cholesterol and phospholipids

• decreases mobility of tails

• membrane less permeable to polar molecule

• thickness of bilayer increases

Lipid movement to other leaflet — scramblase

Enzyme in ER membrane

• catalyzes random flip-flops

Why?

• phospholipids are synthesized in cytosolic leaflet of ER

• to maintain symmetry

Asymmetry of the lipid bilayer

Membranes always have same orientation

• cytosolic leaflet

• noncytosolic leaflet

• Membrane proteins also retain orientation

Lipid movement to other leaflet — Flippases (in? to?)

Enzyme in golgi membrane

• flip-flop of specific phospholipids to cytosolic leaflet

• some can bind cytosolic proteins at plasma membrane (e.g. protein kinase C)

Asymmetry of the lipid bilayer — glycolipids and glycoproteins (in? to?)

• Formed by adding sugar groups to lipids/proteins on luminal face of golgi

• Ends up on plasma membrane or other organelles on noncytosolic face

  • Protects membrane from harsh environments

  • (extracellular, lysosomes, etc)

Lec 14: membrane proteins types overview

Carry out functions in the membrane

Integral

• directly attached to bilayer

• transmembrane/monolayer/lipid covalent attached

Peripheral

• protein attached

• Non-covalent interactions

Integral membrane proteins + extraction methods

Proteins directly attached to lipid bilayer

• inserted into bilayer

• covalently attached to lipid inserted into bilayer

Extraction methods use detergents to denature bilayer

Peripheral membrane proteins

Not inserted into membrane, on either face of membrane (non-covalent interactions)

• bound to other proteins

• bound to lipids

Gentle extraction methods used, lipid bilayer intact

Integral—transmembrane proteins/membrane-spanning proteins

Amphipathic proteins

• Hydrophobic membrane-spanning domains (non-polar side chains)

• Hydrophilic domains in aqueous outside (polar side chains)

Transmembrane protein—single a-helix (single pass)

Membrane spanning helix: ~20 hydrophobic amino acids

• hydrophobic side chains in the membrane

Transmembrane protein—multiple a-helices (multipass)

Multiple a-helices

• hydrophobic side chains on outside of channel

• Hydrophilic side chains on inside

• Forms aqueous channel

Transmembrane proteins—beta-barrel (rolled beta sheet)

Rigid channel, no conformational changes

• hydrophobic side chains on outside

• Hydrophilic side chains on inside

• Forms aqueous channel

Overview of transmembrane protein functions

Each protein has a specific orientation—essential for function

• transporters and channels (sodium/potassium pump)

• anchors (integrins)

• receptors (receptor kinases)

• enzymes (adenylyl cyclase)

How are transmembrane protein structures identified?

1. X-ray crystallography

   1. purify protein crystal

   2. shine x-rays

   3. diffraction pattern

   4. computer program gives structure

2. Hydrophobicity plots

   1. find sections of hydrophobic/hydrophilic amino acids

   2. from N-terminus to C-terminus

   3. above is hydrophobic, below is hydrophilic

   4. look for peaks 20-30 amino acids wide

   5. how many peaks is how many transmembrane domains

Integral—monolayer-associated proteins (a-helix)

Only attached to one layer/leaflet

• amphipathic a-helix

• hydrophobic side chains in leaflet

• hydrophilic side chains on outside

• specialized functions

Integral—monolayer-associated proteins: Sar1 example

Protein in membrane bending to form vesicle

• inserts into cytosolic leaflet

• causes bump in the membrane

• vesicle budding at the ER

Integral—lipid-linked membrane proteins (GPI & lipid anchored)

For specific protein functions/roles, need to be on specific leaflet

.

Protein with GPI anchor

• synthesis in ER lumen

• attach to non-cytosolic face

• end up on outside of cell

.

Protein with lipid anchor

• add to cytosolic face

• end up on inside of cell

Techniques: Extraction of membrane proteins

Mild detergent: denatured bilayer but not protein

• hydrophilic head and hydrophobic tail

• forms micelles in aqueous solution

.

• forms water soluble complexes with amphipathic protein and membrane lipids

Techniques: Studying the properties — liposomes

1. detergent to denature bilayer

2. purify protein

3. add artificial lipids

4. remove detergent

5. form liposome

6. study protein

Techniques: lateral diffusion of membrane proteins (GFP)

Some proteins need to move, some need to be immobile (~range)

Study lateral diffusion — Fluorescence recovery after photobleaching

1. Protein fused to GFP (green-fluorescent protein) or labelled with fluorescent antibody

2. Photobleach area

3. Recovery: Measure rate of diffusion/rate of fluorescence recovery

.

Not anchored, mobile — recovery

Anchored, immobile — no recovery

Lec 15: Permeability of the lipid bilayer overview (can control…)

Artificial bilayer

• impermeable to most water soluble molecules

Cell membrane

• membrane transport proteins to transfer specific molecules — facilitated transport

• can control selectivity and directionality

Movement across the bilayer — without proteins

Permeable (simple diffusion down the concentration gradient)

• small non polar molecules very good, fast

• small uncharged polar not very well

Impermeable (require membrane proteins)

• Larger uncharged polar molecules verrry little

• Ions

What do transport proteins transport

Transmembrane transport protein

• creates protein-lined path across membrane

• transport polar and charged molecules

Transport proteins are selective

• transports specific molecules

Different cell membranes have different proteins

2 main classes of membrane transport proteins

Channel

• selective: size and charge of solute matching

• passive transport

• no conformational changes through open channel

Transporter

• selective: solute fits into binding site

• passive or active transport

• conformational changes for transport

Passive (what kinds?) and active transport

Passive: down concentration gradient (electrochemical gradient if charged)

• simple diffusion

• channel-mediated

• transporter-mediated

Active transport: against gradient

• needs energy

Concentration gradient + Resting membrane potential = Electrochemical gradient (electrical gradient)

Voltage and concentration gradient can work together or against for the net driving force

• Additive or work against

• MOST of the time direction depends on concentration gradient (rate gets affected)

Channel proteins

• Hydrophilic pore across membrane

• Most channel proteins are selective — transient interactions with the channel wall

• ALL Passive transport

• Faster than transporters

Ion channel example (transient recognition) — non-gated vs gated

Selectivity at narrow part of channel

• ion first surrounded by water, then lose

• transient recognition by charged AA in channel

• narrow to select for size

.

Non-gated ion channels — always open

• e.g. K+ leak channels

  • major role in generating resting membrane potential in animal cells

  • negative charge on inside to attract ions

  • but against conc. gradient for transport out of the cell

.

Gated ion channels

• need signal to open channel

• specific ions transported

Types of gated ion channel signals

1. Mechanically-gated: plasma membrane mechanical stress

2. Ligand-gated (extracellular): ligand signal

3. Ligand-gated (intracellular): ligand signal

4. Voltage-gated: change in voltage signal

Transporter proteins

Binds a specific solute

• goes through conformational change to transport solute across membrane

Rate of transport: channel vs transporter

Channel transport rate increases as concentration difference increases

Transporter transport rate has a maximum rate due to all binding sites completely saturated

(channel proteins transport faster)

Transporter proteins: Uniport (direction is….)

Uniport: one solute

• passive transport down electrochemical gradient

• direction of transport is reversible

• random conformational changes, just happens to transport

E.g. GLUT uniporter

• glucose is the only uncharged

Types of active transport — needs energy (3 kinds)

1. Gradient driven pump

2. ATP driven pump (ATPases)

3. Light-driven pump (bacteria)

Active transprt: Gradient driven pumps — Symport and anitiport

Symports: two molecules moving in same direction

Antiport: two molecules moving in opposite direction

• Free energy from one solute moving down its electrochemical gradient

• Powers transport of other solute moving against its electrochemical gradient

Symport example: Na+ —glucose symporter

Na+ down its electrochemical gradient provides energy to move glucose against concentration gradient

• random oscillations between conformations

• BUT conformational changes only happen if both sites occupied (cooperative binding) or both sites empty

  • occluded-empty or occluded-occupied

  • then open up again randomly

.

Sits open with Na+ until glucose binds

• if open to the other side, sodium and glucose released

Antiport example: Na+—H+ exchanger (transported to?)

Na+ down its electrochemical gradient provides energy to move H+ against its

electrochemical gradient

• Cytosolic pH needs to be regulated for best enzyme function (pH~7.2)

• But excess H+ occurs in cytosol

  • from acid forming reactions, or leaks out of lysosome (pH~5)

Transporters maintain cytosolic pH

.

e.g. Na+ — H+ exchanger in the plasma membrane

• Excess H+ in cytosol

• transporter activity increases

• H+ transported to extracellular space

How is the Na+ electrochemical gradient maintained?

Symport, Antiport needs Na+ electrochemical gradient to provide energy

• but continued action could equalize Na+ gradient

How is it maintained in animal cells? Na+ K+ pump (plasma membrane ATP driven pump)

ATP-driven pumps

Active transport, use energy from ATP hydrolysis to transport solutes

Types of ATP-driven pumps — P type pump (example?)

Use ATP

• Phosphorylated during pumping cycle

• Many types (transport ions)

• AND flippases (transport phospholipids)

ATP-driven pumps: ① P-type pump (Na/K pump) (extracellular and cytosol conc?)

Animal plasma membrane

Na+ and K+ moved against their electrochemical gradients

• 3 Na+ out

• 2 K+ in

• more positive out than in

(Na+ gradient available for gradient driven transport)

.

Na+ high in extracellular

K+ high in cytosol

Pumping cycle of the Na/K pump

Has to go through these sequences

• 3 sodium ions bind to pump (open to cytosol)

• ATP hydrolysis —→ phosphorylation —→ change comformation, sodium out

• 2 potassium ions bind (open to extracellular

• dephosphorylate —→ change conformation back, potassium out

(then start again)

(phosphorylation and dephosphorylation powers the pump)

• if any stage was blocked, stop working

P-type pumps and electrochemical gradients — animal vs plant

Function in generating and maintaining electrochemical gradients

.

Animal — Na+/K+ pump, animal cell plasma membrane

.

Plant — H+ pump, plant cell plasma membrane (doesnt have Na/K pump)

Types of ATP driven pumps — other than P (what does V do?)

2. ABC transporter

   • use 2 ATP hydrolysis, pump small molecules

   • pump toxins out of the cell (bad for cancer treatment)

3. V-type proton pump

   • pumps H+ into organelles to acidify the lumen

F-type ATP synthase

Structurally related to V-type proton pump, but opposite mode of action

• uses H+ gradient to drive the synthesis of ATP

• in mitochondria, chloroplasts, bacteria

Transport proteins regulate critical cellular processes (Transporter can work together)

• Transcellular transport of glucose by transporters

• Generation of membrane potentials

e.g. GLUT uniporter (passive), Na+ glucose symporter, Na+K+ pump

Transporters work together to transfer glucose from the intestine to the bloodstream

Glucose taken up from gut

• Na+/glucose symporter on the apical domain villi

• (against gradient, ACTIVE TRANSPORT)

Transported through cell

Transported to bloodstream via GLUT uniporter

• on basalateral domain

• (down gradient, PASSIVE TRANSPORT)

Na+ K+ pump on basal domain to maintain membrane potential

.

(low glucose in gut, high in cytosol of epithelial cell, low in extracellular fluid)

Transcellular Transport of Glucose Requires the Asymmetric Distribution of Membrane Proteins

Intestinal epithelial cells

• Transport proteins restricted to certain places by tight junctions (creates boundary, seals)

  • Apical membrane — Na+-glucose symporter

  • Basolateral plasma membrane — GLUT2 uniporter

  • Na+-K+ pump

Membrane potential (pumps and?)

Difference in electrical charge on two sides of membrane

• Used by gradient-driven pumps to carry out active transport (Symport, Antiport)

• Electrical signaling

Generation of Membrane Potential (animal cells)

1. K+ Leak channel (to extracellular)

   • major role in membrane potential

   • passive transporter

   • voltage gradient opposite to concentration gradient, creates positive charge

   • will reach equilibrium — no net movement

.

1. Na+-K+ pump (to extracellular)

   • ~10% of membrane potential

   • Na+ gradient with low cytosolic [Na+]

   • K+ gradient with high cytosolic [K+]

   • 3Na:2K, Net 1+ ion out

Generation of Membrane Potential (animal cells) — what is resting membrane potential?

Net result:

• Bit more positive on outside (Na+, K+)

• Bit more negative on inside (Cl- and fixed anions)

Equilibrium = resting membrane potential

(animal cells: vary from -20 mV to -200 mV)

Generation of Membrane Potential (plant cells) (used for?)

Plasma membrane P-type pump (to extracellular or lumen)

• H+ pump

• generates H+ electrochemical gradient = membrane potential

• -120 to -160 mV (in cytosol)

.

Used for:

• Used by gradient-driven pumps to carry out

• active transport (e.g. H+ driven symport)

• Electrical signaling

• Regulate pH

Lec 17: Intracellular compartments

Mitochondrion, Golgi, ER, vesicles, endosome, peroxisomes…

Intracellular Compartments - volumes

Cytosol takes up half the cell volume

Volumes will differ for different cell types

Intracellular compartments — membranes

Plasma membrane very small portion of the cell membranes

Rough and smooth ER take up 50-60%

• Rough ER — membrane bound ribosomes, synthesis of soluble and transmembrane proteins

• Smooth ER — phospholipid synthesis, detox

Organelle definition

A discrete structure or subcompartment of the a eukaryotic cell that is specialized to do a particular function

Organelle types

Membrane-enclosed

• Nucleus

• ER

• Golgi

Not membrane-bound

• Nucleolus

• Centrosome

• biomolecular condensates

Protein sorting — simple

Proteins are nuclear encoded (made from genes)

mRNA arrives in cytoplasm — translation starts on cytosolic ribosomes

• Cytosolic protein: no sorting signal, defualt location is cytosol

• Sorted proteins: have sorting signal, signal sequence

(can be modified experimentally to identify the types of signals)

How are specific proteins sorted to different organelles? (signal sequence, sorting receptors)

Sorted by signal sequence

• Part of AA sequence

• Specifies desination — nucleus, mitochondria, ER, peroxisomes,

  etc

• Recognized by sorting receptors that take proteins to the destination

  • Signals are very different

Protein sorting: post vs co-translational

Post-translational

• Proteins nuclear-encoded

• Fully synthesized in cytosol before sorting

• Can be folded or unfolded

Co-translational sorting

• Proteins nuclear-encoded

• ER signal sequence

• associated with ER during protein synthesis in the cytosol

Proteins for nucleus (whats the signal called?)

Post-translational

• Nuclear-encoded

• Fully synthesized

• Folded

.

• Targeted by signal sequence for import into nucleus (nuclear localization signal)

• Recognized by sorting receptor

• Transported through nuclear pore

• Detached

Example

• transcription factors required in the nucleus

Mutation of nuclear localization signal

If mutated, not recognized by sorting receptor — stays in the cytosol

Proteins for Peroxisomes (through the?)

Post-translational sorting

• Nuclear-encoded

• Fully synthesized

• Folded

.

• Targeted by sorting receptor for import into peroxisome

• Transported into peroxisomes through transmembrane protein complex

.

e.g Enzymes for oxidative reactions

Proteins for Mitochondria & chloroplast

Post-translational sorting

• Nuclear encoded

• Fully synthesized

• Unfolded

.

• Targeted by signal sequence

Proteins for Mitochondria & chloroplast — continued

Mitochondria and chloroplasts have own genomes and ribosomes

• But most proteins are nuclear encoded

• Unfolded to transport through transmembrane proteins

• Asociated with cytosolic hsp70 chaperone proteins

In matrix — mitochondrial hsp70 fold protein into shape

• Signal cleaved

Proteins for the ER (signal sequence is hydro?)

Co-translational sorting

• Nuclear encoded

• ER signal sequence — hydrophobic

  • associated with ER during protein synthesis in the cytosol

Proteins for the ER — Why sort to ER?

Entry point to the endomembrane system

Endoplasmic Reticulum (soluble and transmembrane proteins vs phospholipids)

Rough ER (with membrane-bound ribosomes)

• synthesis of soluble and transmembrane proteins for the endomembrane

.

Smooth ER

• phospholipid synthesis, detoxification

Sorting Proteins to the ER

mRNA arrives at cytoplasm

• Translation starts on ribosomes in cytosol

ER signal sequence

• Insertion of protein into ER starts as translation continued

.

• Soluble proteins

• Transmembrane proteins

Co-translational Translocation: Soluble protein (SRP, SRP receptor, Translocon, Signal peptidase)

1. Translation start, N-terminal ER signal sequence emerges

2. Recognized by SRP, elongation stopped

3. SRP-ribosome complex — to SRP receptor — to translocon

4. Translocon opens

5. Protein synthesis resumes with protein transfer into ER lumen

6. Signal peptidase cleaves ER signal sequence (hydrophobic — in bilayer)

7. Protein released into ER lumen

8. Translocon closes

Destination: lumen of endomembrane organelles or extracellular secretion

Co-translational Translocation: Transmembrane protein

• SRP-ribosome complex

• Stop-transfer sequence (hydrophobic a-helix) enters translocon

• Protein transfer stops

• Transmembrane domain released into bilayer

• Signal peptidase cleaves

• Translocon closes

• protein synthesis completed

Destination: membrane of endomembrane organelle or plasma membrane

ER signal sequence types (N terminal & internal)

N-terminal ER signal sequence

• At N-terminus

• hydrophobic AA

• removed by signal peptidase

.

Internal ER signal sequence

• start-transfer sequence

• hydrophobic AA

• not removed — becomes membrane spanning a-helix

Co-translational Translocation: Transmembrane protein — multipass

• Start-transfer emerges, pause ribosome, move to ER membrane

• Transports through membrane until Stop-transfer sequence enters translocon

• Both are released into bilayer — membrane spanning a-helices

• Translocon closes, translation finishes

Number of transmembrane domains determines whether terminuses are on the same side or opposite sides

Intracellular compartments are dynamic

Endomembrane system

• ER

• Golgi

• Endosomes

• Lysosomes

.

Intracellular compartments exchange components

• Proteins (soluble and membrane)

• Lipids — fused into destination membrane

Pathways of the endomembrane system

Secretory pathway

• proteins and lipids from the ER

• ER to outside (exocytosis)

• ER to lysosomes (endosomes)

.

Endocytic pathway

• contents move into cell (endocytosis)

• degredation

Retrieval pathway

• Retrieval of lipids and selected proteins for reuse

Exocytosis and Endocytosis

Exocytosis:

• vesicle contents delivered to extracellular space

• vesicle membrane becomes part of the plasma membrane

Endocytosis:

• vesicle contents come from extracellular space

• vesicle membrane buds from plasma membrane

Vesicular transport (cargo and specificity ddd?)

Vesicle is:

• small membrane-enclosed organelle in cytoplasm

• Soluble proteins + membrane proteins = cargo

  • receptors in membrane select cargo

  • or diffusion into vesicle

  • receptors can get recycled

Specificity from:

• docking proteins bringing vesicle to target membrane

Secretory pathway — constitutive exocytosis pathway (proteins & lipids)

• In all eukaryotic cells

• continual delivery of proteins (transmembrane and soluble) and lipids to plasma membrane

• not regulated

Secretory pathway — Regulated exocytosis pathway

• regulated secretion in specialized cells

• specialized secretory vesicles

• need extracellular signal before vesicle fuses with plasma membrane

e.g. insulin and blood glucose levels

Path of secreted proteins from translation to plasma membrane

• Translation starts in cytosol

• Co-translational translocation at ER

• Secreted protein through secretory pathway

Path of a transmembrane protein from translation to plasma membrane

• Translation starts on cytosolic ribosomes

• Co-translational translocation at ER

• Transmembrane protein through secretory pathway — fused with plasma membrane

Maintenance of membrane protein asymmetry

• Membrane has orientation

• Protein asymmetry is maintained through vesicular transport

Golgi apparatus (receive….and?) (animal vs plant)

• recieves proteins and lipids from ER, modifies them, then dispatches them to other destinations in the cell

• Stacks of enclosed sacs

Animal cells has one large golgi

Plant cells have many small golgi

Protein glycosylation

Starts in the ER

• single type of oligosaccharide is attached to many proteins on lumen side

.

Complex oligosaccharide processing occurs in golgi apparatus

• different enzymes in each cisterna

• glycosylation modifications for proteins and lipids

Endocytic pathway: endosomes and lysosomes

Membrane bound organelles containing meterial digested by endocytosis

Endocytic pathway: endosomes and lysosomes: endocytic vesicles

• Fuse with early endosomes and with eachother to form early endosomes

• ingested material sorted, stays in endosomes or recycled to plasma membrane

Endocytic pathway: endosomes and lysosomes: gradual maturation of early endosomes into late endosomes

1. moves to nucleus, stops recycling material to plasma membrane

2. hydrolases and proton pump delivered from golgi by vesicles

3. protons pumped into endosome — acidification

4. hydrolases become more active

Maturation of late endosomes into lysosomes

1. hydrolases and proton pump continue delivered

2. pH continues to drop, hydrolase digest

3. late endosomes fuse with each other and with lysosomes

Lysosome - final detination for endocytosed material being digested

other pathways available — autophagy

Lysosomes are the main site of intracellular digestion (enzymes, membrane, transport)

Contains 40 types of hydrolytic enzymes

• acid hydrolases

• Lysosomes are acidified by proton pump

.

Membrane-bound organelle

• protects cell from digestion

• glyosylated proteins to protect from proteases

.

Transport proteins in lysosomal membrane

• transfer digested monomers to cytosol

Cytoskeleton

Network of protein filaments, Highly dynamic

Many important functions