Lecture 36: Membranes and Membrane Proteins11/26/11 4:25 PM

Cell membranes act as selective barriers

Prevent molecules from mixing

Three roles of plasma membrane

Receiving information (signaling)

Import/export (transportation)

Motility/cell growth

Membranes enclose many different compartments in a eukaryotic cell:

Nucleus (2x)

Mitochondria (2x)

ER, vesicles, golgi apparatus, lysosome, peroxisome

The Lipid Bilayer

Two-dimensional fluid

Fluidity depends on composition

Lipid bilayer is asymmetrical

Lipid asymmetry is generated inside the cell

Hydrophilic head, hydrophobic tail

The more unsaturated the tails are, fluidity is increased

Phosphatidylcholine is the most common phospholipid in cell

membranes

There are different types of membrane lipids and all are amphipathic

Hydrophilic molecules attract water, like dissolves in like

Hydrophobic molecules avoid water

Fats are hydrophobic, phospholipids are amphipathic (and form a

bilayer in water)

Pure phospholipids can form closed, spherical liposomes

Phospholipids can move

o Lateral

o Flexion

o Rotation

o Flip-flop

Fluidity depends on composition

o Cholesterol stiffens

o Low temperature, less unsaturation, long tails all reduce

fluidity.

Phospho and glycolipids are distributed asymmetrically in the plasma

membrane

o Glycolipids found on outside

o Phosphatidyl-serine, inositol, ethanolamine found on inside

usually.

Flippases transfer phospholipids to other side of membrane

New membranes are synthesized from ER.

o Form vesicles which fuse with other membranes

Membrane Proteins

Polypeptide chain usually crosses the bilayer as an α-helix

Proteins can be solubilized in detergents and purified

Plasma membrane is reinforced by the cell cortex

Cell surface is coated with carbohydrate

Cells can restrict movement of membrane proteins

Functions

o Transporters (ie Na pump)

o Anchors (integrins)

o Receptors (platelet-derived growth factor receptor)

o Enzymes (adenyl cyclase)

50% of mass of plasma membranes, 50 times more lipid than protein

molecules.

Different ways of associating with membrane:

o Alpha helix, beta pleated sheets, transmembrane, lipid linked

o Can be peripheral (protein attached)

Folded up proteins traverse membrane easier because the polar

backbone is exposed

Multiple alpha helixes form a hydrophilic pore

Porin proteins form water-filled channels in the outer membrane of a

bacterium

o Formed by 16 strands of β-sheets

o Allows passage of ions and nutrients across outer membranes

of some bacteria and of mitochondria

Membranes are disrupted by detergents such as SDS and Triton X-100

o Have only one tail

Bacteriorhodopsin acts as a proton pump powered by light, drives ATP

synthase

Plasma membrane reinforced by cell cortex – imparts shape and

function

o Spectrin meshwork forms the cell cortex in red blood cells

Eukaryotic cells are sugar coated

o Absorb water for lubrication

o Cell-cell recognition

o Protect cell from physical, chemical, enzymatic damage

o Recognition of cell surface carb on neutrophils mediates

migration in infection

Movement can be restricted by cells

o Tethering to cell cortex, extracellular matrix, proteins on

surface of another cell, or by barriers of diffusion like tight

junctions.

Lecture 37

I. General Principles of Cell Signaling

Can act over long or short range

Each cell responds to limited set of signals

Signals relayed via intracellular signaling pathways

Nitric oxide crosses plasma membrane and activates intracellular

enzymes directly

Some hormones cross plasma membrane and bind to intracellular

receptors

There are three classes of cell surface receptors

Ion channel-linked receptors convert chemical into electrical signals

Intracellular signaling proteins act as molecular switches

Origins in unicellular organisms

o Yeast shows single cell-cell communication

o Two mating types, a and α plus a secreted mating factor

signal

Signals transduction: conversion of one type of signal into another

o Extracellular -> intracellular

4 ways animal cells signal

o Endocrine

o Paracrine

o Neuronal

o Contact-dependent

Lateral inhibition: Unspecified epithelial cells, one cell is

dedicated to becoming a nerve cell and inhibits

surrounding cells by Delta-Notch signaling.

One signal molecule can induce different responses in different cells

o ie: acetylcholine: (time scale is seconds to minutes)

In heart muscle cells, causes decreased rate and force

of contraction.

In salivary gland cells, causes secretion.

In skeletal muscle cells, causes contraction.

An animal cell depends on multiple extracellular signals

Extracellular signal molecules can alter activity of diverse cell proteins

which in turn alter cellular behavior

o The intracellular signaling proteins are involved in a signaling

cascade which ultimately reach the target proteins for altered

behavior like metabolism, gene expression, and cell shape or

movement.

Cellular signaling cascades can follow a complex path

o Primary transduction, relay, amplification, or branching to

different targets.

Extracellular signal molecules can either bind to cell surface receptors

or to intracellular enzymes or receptors (like nitric oxide)

o Nitric oxide is a product of nitroglycerin which is taken to

relax smooth muscle cells.

Triggers smooth muscle relaxation in blood-vessel wall

Steroid hormones bind intracellular receptors that act as gene

regulatory proteins

o Cross plasma membrane, like NO

o Cholesterol does not cross membrane, rather inserts IN

membrane.

o Cortisol acts by activating a gene regulatory protein

Most signal molecules bind to receptor proteins on the target cell

surface

o Extracellular domains are the cell surface receptor

o Three basic classes:

Ion channel linked -> nervous system, muscle

G-protein linked -> all cells

Enzyme-linked -> all cells

Many intracellular signaling proteins act as molecular switches

o Signaling by phosphorylation

Signal in by phosphorylation, off by phosphatase

inactivation.

o Signaling by GTP-binding protein

GTP binds to G-protein, turning it on.

GTP hydrolysis inactivates by removing P.

II. G-protein-linked Receptors

Stimulation of G-protein linked receptors

G proteins can regulate ion channels

G proteins can activate membrane bound enzymes

Cyclic AMP pathway can activate downstream genes

Inositol phospholipid pathway triggers rise in Ca

Ca signal triggers many biological processes

Intracellular signaling cascades can achieve astonishing speed (ie

photoreceptors in the eye)

All G-protein linked receptors possess a similar structure

o 7 transmembrane protein

o Ligand binds to extracellular binding domain

o Cytoplasmic domain which binds to G-protein

o Tetramer is active, GDP can dissociate, GTP can bind, and

then complex dissociates into two activated parts.

o The alpha subunit switches itself off by hydrolyzing

bound GTP

G proteins couple receptor activation to opening of cardiomyocyte K

channels

o Acetylcholine binds to G protein linked receptor

o Beta gamma complex binds to closed K channel to open it

o Alpha subunit is inactivated (by hydrolysis) and inactive

complex reassociates with betta gamma complex to close K

channel.

Enzymes activated by G proteins catalyze synthesis of intracellular

second messengers

o Alpha subunit activates adenylyl cyclase which makes lots of

cylic AMP.

o Cyclic AMP concentration rises rapidly in response to

neurotransmitter serotonin

o Cyclic AMP is synthesized by adenylyl cyclase, degraded by

cAMP phosphodiesterase

Extracellular signals can act rapidly or slowly

Rise in intracellular cyclic AMP can activate gene transcription through

protein kinase A

o Translocates through nuclear pore, into nucleus,

phosphorylates gene regulatory protein to activate target

gene.

Membrane bound phospholipase C activates two small messenger

molecules: IP3, DAG

o Phospholipase C activated by alpha subunit, splits inositol

phospholipid into IP3 and DAG

o IP3 opens Ca channel in ER, Ca is released and works with

DAG to activate Protein Kinase C.

Fertilization of an egg by sperm triggers a rapid increase in cytosolic Ca

o Other processes triggered by Ca signal:

Sperm entry -> embryonic development

Skeletal muscle -> contraction

Nerve cells -> secretion

Calcium/Calmodulin complex are what bind to proteins.

A rod photoreceptor cell from the retina is exquisitely sensitive to light

o G protein linked light receptor activates G protein

transducing, activated alpha subunit causes Na channels to

close.

o Light induced signaling cascade in rod photoreceptors greatly

amplifies light signals.

III. Enzyme linked receptors

Activated receptor tyrosine kinases assemble a complex of intracellular

signaling proteins

o Ligand brings two tyrosine kinase domains together,

phosphorylated to activate. Intracellular signaling proteins

bind to phosphorylated tyrosines.

o Activated complex includes Ras-activating protein, which is

anchored in membrane, transmits signal downstream.

Ras is monomeric GTP-binding protein, not a trimeric G

protein, but resembles the alpha subunit and functions

as a molecular switch.

30% of cancers arise from mutations in Ras.

Ras activates a MAP-kinase phosphorylation cascade

Some enzyme-linked receptors activate a fast track to the nucleus

Protein kinase networks integrate info to control complex cell behaviors

Multicellularity and cell communication evolved independently in plants

and animals

Cytokine receptors are associated with cytoplasmic tyrosine kinases

o JAK kinases phosphorylate receptor which recruits cytoplasmic

proteins.

TGF-beta/BMP receptors activate gene regulatory proteins directly at

the plasma membrane

Signaling pathways can be highly interconnected: cross-talk

Lecture 38

General introduction:

Membrane enclosed organelles are distributed throughout the

cytoplasm

o Thousands of different reactions occur simultaneously, are

partitioned

o Cytosol is 54% of cell

o Mitochondria is 22% of cells

o ER is 12% of cell (1 per cell)

Nuclear membrane and ER may have evolved at the same time through

invagination of plasma membrane.

Mitochondria are thought to have originated from aerobic prokaryote

being engulfed by a larger anaerobic eukaryotic cell -> has it’s own

genome.

Nucleus is a double membrane organelle

o Encloses nuclear DNA, defines nuclear compartment and

contains most of the genetic information.

o Export, import through nuclear pore complex

Contains about 100 proteins, two way gate, export of

mRNA, and ribosome subunits.

Import of proteins requires a signal sequence called the

nuclear localization signal

Requires energy (GTP) and special chaperone proteins

Export of RNA from nucleus – RNA molecules are made

in the nucleus and exported to the cytoplasm as

processed mRNA

One Endoplasmic Reticulum

o System of interconnected sacs and tubes of membrane

o Extend throughout most of cell

o Major site of new membrane (lipid) synthesis

o With ribosomes on cytosolic side = rough ER

o Without ribosomes = smooth ER

o Most extensive network membrane in eukaryotic cells

Golgi apparatus

o Flattened sacs called cisternae which are piled like stacks of

plates

o Usually near nucleus

o Two faces:

Cis face adjacent to ER

Trans face towards plasma membrane (where post

translational modification occurs)

o Receives proteins and lipids

o Site of modification of proteins and lipids

o Dispatches proteins and lipids to final destinations

o Transport vesicles bud off

Other membrane enclosed organelles

o Endosomes – small membrane enclosed organelles that sort

ingested molecules in endocytosed materials. Passed to

lysosomes or recycled back to the plasma membrane.

o Lysosomes – small sacs containing digestive enzymes that

degrade organelles, macromolecules, and particles taken in

by endocytosis. “garbage disposal of the cell.” Ph about 7.2

o Peroxisomes – small membrane enclosed organelle containing

oxidative enzymes that break down lipids and destroy toxic

molecules

Protein transport

o Multiple modes of protein transport (import and export)

o Three mechanisms

Transport through nuclear pores: protein with nuclear

localization signal enter through pores

Across membranes: proteins moving from cytosol into

ER, mitochondria and peroxisomes transported across

organelle membrane by protein translocators

By vesicles: from ER onward and from one

endomembrane compartment to another ferried by

transport vesicles

Protein sorting signals

o Specific amino acid sequence

o Directs protein to organelle

o Proteins without signals remain in cytosol

o Signal sequences direct proteins to different compoartments

Continuous stretch of AA usually 15-20 residues in

length

Usually removed after the protein reaches destination

Organelles and signal sequences:

ER import rich in V A L I and retendtion KDEL

Mitochondria rich in R

Nucleus PPKKKRKV

Peroxisomes SKL

o Signal sequences are both necessary and sufficient to direct

protein to organelles

ER: entry point for protein distribution

o Proteins destined for golgi, lysosomes, endosomes and cell

surfaces first enter ER from cytosol

o Once inside ER or membrane, proteins do not reenter cytosol

o Water soluble proteins are completely translocated across ER

membrane and released into ER lumen

o Transmembrane proteins only partially translocated across ER

membrane and become embedded

Vesicular transport

o Entry into ER

o To golgi apparatus

o From er -> golgi -> other by continuous budding, fusion of

transport vesicles

o Vesicle transport provides routes of communication

Protein transport: quality control

o Most proteins that enter ER are destined for other locations

o Exit from the ER is highly selective: improperly modified and

or folded proteins are retained in lumen; dimeric or multimeric

proteins that fail to assemble are also retained

Exocytosis

o Constitutive: newly synthesized proteins, lipids, and carbs

delivered from ER via golgi to subcellular locations,

extracellularly to ECM via transport vesicles.

Lipids and proteins supplied to plasma membrane

Proteins secreted into ECM or onto the cell surface

o Regulated

Specialized secretory cells synthesize high levels of

proteins such as hormones or digestive enzymes that

are stored in secretory vesicles for subsequent release

Vesicles bud off from trans golgi network and

accumulate adjacent to plasma membrane until

mobilized by extracellular signal

Endocytosis

o Pinocytosis (drinking)

Internalizes plasma membrane: as much membrane is

added to cell surface by exocytosis as is removed by

endocytosis – total surface area and volume remain

unchanged.

Mainly carried out by transport vesicles: deliver

extracellular fluid and solutes to endosomes; fluid

intake is balanced by fluid loss during exocytosis

o Phagocytosis (eating)

Specialized cells only

o

Lecture 39: Cytoskeleton

Roles of cytoskeletal filaments:

Intermediate – cell structure against mechanical stress

Microtubules – intracellular transport, railroad of cell

Actin – membrane mobility; cell movement

Intermediate filaments:

10 nm in diameter

Rope like structure composed of long polypeptides twisted together

Associated with cell junctions

Mechanical strength, cell shape, cell-cell contacts, and structure for

nuclear envelope

Monomers -> dimer -> tetramer -> 8 tetramers make one ropelike

filament

Different proteins:

o Epithelia – keratins

o Connective tissue, muscles, neuroglial cells – vimentin

o Nerve cells – neurofilaments

o Nuclear envelope in animal cells – nuclear lamins

Mutation in keratin genes = epidermolysis bullosa simplex

Networks of filaments connect across desmosomes in epithelia

Microtubules:

25 nm wide

Hollow, made of α and β tubulin anchored to γ tubulin

Have polarity – gives directionality

“Dynamic instability”: built or disassembled as needed

o Zip up to grow

o Unravel and tubulin molecules fall off if not needed

o This is done by GTP since tubulin are GTPases

GTPases are the cell’s timers

High energy phosphate bond. Molecules with GTPases

hydrolyze that bond, leaving GDP

GTP between α and β tubulin molecules makes them

straighter, so they pack better. GTP hydrolysis makes

them kinked, so they fall off.

Organize cell organelles and control traffic of vesicles

Roles in interphase cell, dividing cell, ciliated cells, flagella.

The centrosome

o Centriles inside of centrosome, nobody knows what they do

o Centrosome is an envelope of tubulin where microtubules

extend out with plus end out.

Structure:

o α and β tubulin strands

Stabilizing or destabilizing MTs

o Microtubule associated proteins (MAPs)

Bind to free ends of MTs and stabilize ends selectively

to polarize a cell

o Drugs can be used to change MT stability

Colchicine binds free tubulin to prevent polymerization;

MTs disintegrate and mitosis stops

Taxol prevents loss of subunits from MTs; MTs become

“frozen” in place and mitosis stops.

MT organized transport

o Anterograde transport, retrograde transport.

Motor proteins use ATP to power transport along the MT railroad

o Kinesin and dynein are dimers that walk along microtubule.

o One ATP is used per step.

Cilia and flagella are made of MTs

Actin Filaments:

Control cell movement

Found in:

o Epithelial cell microvilli

o Stress fibers in cultured cells

o Leading edge lamellipodia

o Contractile ring in dividing cells – cytokinesis

Actin polymerization requires ATP

o Free G-actin monomers use ATP to become F-actin to form

filaments. To uncoil, hydrolyze ATP and fall apart.

Actin dynamics provide force for membrane movement

o ARP complex create branches

o Depolymerizing protein promotes ATP hydrolysis

o Capping proteins cap the ends and stabilize ATP bound

monomer, stabilizing leading edge.

Actin binding proteins link actin fibers to the membrane and other

cellular components

Integrins link actin to focal adhesions

o Binds to extracellular structures, messages to actin.

Cells move by actin crawling (dynamics)

Axon growth cone crawling

Rho family GTPases control actin dynamics

o RhoA causes stress fibers

Stabilize actin filaments

Induces myosin phosphorylation and thus contractility

o Cdc432 causes filopodia extension

Promotes actin nucleating by ARP complexes

o Rac promotes lamellipodia extension

Promotes actin nucleation, but also uncapping to allow

more sites of nucleation

o Cell surface receptors modulate Rho family activity

Attractive cues activate Rac and Cdc42 on area of

growth cone

Repulsive cues activate RhoA

Growth cone turns

Myosins: actin motor proteins

o Head, neck, tail

o Tails link up together

o Work as dimers

o Moves membranes or cell components

Muscle contraction by actin and myosin

o Myosin heads climb up actin filament

o Z disks move together, muscle contracts