Module 1: Transport of Small Molecules and Proteins

plasma membrane

defines the cell and separates the cytosol from the extracellular environment

membrane: phospholipid bilayer (2D fluid)

fluidity: olive oil like

• noncovalent interactions between phospholipids, and between phospholipids and proteins provide membrane integrity and resilience

• individual phospholipids spin and diffuse laterally within the plane of the membrane

barrier: hydrophobic core prevents unassisted movement of water-soluble substance from one side to the other

protein: membrane proteins provide each cellular membrane its unique set of function

• integral membrane proteins (transmembrane proteins) span bilayer and often form dimer and high-order oligomers

• lipid-anchored proteins tethered to one leaflet by a covalently attached hydrocarbon chain

• peripheral protein associated primarily by specific noncovalent interactions with integral membrane proteins or membrane lipids

eukaryotic cellular membranes are dynamic structures

membrane fluidity and flexibility

• enables organelles to assume their typical shapes

• provides dynamic property that enables membrane budding and fusion

HIV-infected cell plasma membrane

virus core - enveloped by region of cell plasma membrane that contains specific viral proteins

HIV particles - bud from plasma membrane

Golgi complex

stacked membranes with budding vesicles involved in intracellular trafficking

amphipathic

________ phospholipids spontaneously form bilayer with hydrophilic faces and a hydrophobic core

• biological membranes

  • vary in lipid composition

  • impermeable to water-soluble molecules and ions

  • have viscous consistency with fluidlike properties

membrane phospholipids

amphipathic molecules - ends have different chemical properties:

• hydrophobic fatty acid-based hydrocarbon “tail”

• hydrophilic polar “head” which interacts with water molecules

erythrocyte membrane

stain interaction with hydrophilic heads and not hydrophobic tails yields characteristic “railroad track” appearance

true

T/F: Phospholipids structure form spontaneously, driven by behavior of hydrophilic and hydrophobic end exposure to water

nonpolar tails

close packing stabilized by van der Waals and hydrophobic effects interactions between the hydrocarbon chains

polar head groups

ionic and hydrogen bonds stabilize interactions with each other and with water

• face outward to shield the hydrophobic fatty acyl tails from water

• hydrophobic effect and van der Waals interactions between the fatty acyl tails drive the assembly of the bilayer

spherical micelle

phospholipid structure in water

hydrophobic interior composed entirely of fatty acyl chains

• will not accommodate biomembrane phospholipids with 2 tails

• detergents and soaps with less bulky tails form micelles

spherical liposome

phospholipid structure in water

• phospholipid bilayer surrounding an aqueous compartment

cubic phase

unnatural highly regular recurring structure

helped formation of membrane protein crystals for structure determination

true

T/F: Cellular membranes have 2 faces, the cytosolic face and the exoplasmic face

true

T/F: The internal aqueous space is topologically equivalent to the outside of the cell

nucleus, mitochondrion, and chloroplast organelles

enclosed by two membranes separated by small intermembrane space

exoplasmic faces of the inner and outer membranes border the intermembrane space

endocytosis

plasma membrane segment buds inward toward the cytosol and eventually pinches off a separate vesicle

• cytosolic face - remains facing cytosol

• exoplasmic face - faces vesicle lumen

exocytosis

an intracellular vesicle fuses with the plasma membrane

• vesicle lumen connects with the extracellular medium

• cytoplasmic face remains facing cytoplasm

true

T/F: Membrane-spanning proteins retain asymmetric orientation during vesicle budding and fusion; same protein segment(s) always faces the cytosol

endosymbiont hypothesis

many lines of evidence that mitochondria and chloroplasts evolved from eubacteria engulfed into ancestral cells containing a eukaryotic nucleus

true

T/F: In organelles with two membranes the exoplasmic surface faces the space between the membranes

endocytosis of bacterium by an ancestral eukaryotic cell

1) eubacterium endocytosed with 2 membranes

2) becomes organelle with 2 membranes

• outer membrane = derived from eukaryotic PM

• inner membrane = originally the bacterial PM

• inner membrane proteins would retain orientation

3) budding of vesicles from the inner chloroplast membrane vesicle; generates thylakoid membranes with the F1 subunit (ATP synthase) facing the chloroplast stoma

erythrocyte cell

discoid - flexible shape required for squeezing through blood capillaries with smaller diameters

shape defects cause cell lysis (anemias)

ciliated cells in the trachea

cilia extensions of the PM contain microtubules and motor proteins that enable them to produce patterns of shape changes that move materials across epithelial surfaces or propel cell motility

an immotile primary cilium plays key roles in cell signaling

three classes of lipids

differ in structure, abundance, and function

1. phosphoglycerides (phospholipids)

2. sphingolipids (phospholipids or glycolipids)

3. sterols

phosphoglycerides

most abundant in biomembrane

• glycerol backbone

• tails - 2 esterified hydrophobic fatty acyl chains

  • usually 16C-18C

  • vary in saturation (saturated/unsaturated or bonds/double bonds)

• head - a polar group esterified to the phosphate (4 types)

  • phosphatidylcholine (PC)

  • phosphatidylethanolamine (PE)

  • phosphatidylserine (PS)

  • phosphatidylinositol (PI)

sphingolipids

derivatives of sphingosine (an amino alcohol with a long hydrocarbon chain)

• various fatty acyl chains connected by an amide bond

• some are glycolipids that contain a single sugar residue or branched oligosaccharide attached to the sphingosine backbone (ex: Glucosylcerebroside GlcCer has a glucose head group)

sphingomyelins (SM)

contain a phosphocholine head group

sterols

membrane components - animals (cholesterol), fungi (ergosterol), and plants (stigmasterol)

amphipathic structure

• head group = single polar -OH

• tail = conjugated four-ring hydrocarbon and short hydrocarbon chain

very hydrophobic

cannot form bilayers on its own, but it intercalates into biomembrane

temperature influence on biomembrane

COLD

• gel-like consistency

• below the phase transition temperature fatty acyl chains are in a gel-like (crystalline) state

HEAT

• fluid-like consistency

• above the phase transition temperature fatty acyl chains are in rapid motion

• heat disorders nonpolar tails induces a transition from a gel to a fluid within a temperature range of only a few degrees

• chain disorder increases bilayer thickness

lipid composition

within same cell, different membranes have different lipid compositions

• ex: Golgi membranes contain more sphingomyelin than ER membranes

different types of cells have membranes with different _________

• ex: PM of intestinal cells contains more sphingolipids and less phosphoglycerides then most

fluid

A more _______ state is favored by lipids with short fatty acyl chains (like phosphoglycerides

gel-like

A more _______ state is favored by longer more saturated fatty acyl chains that pack tightly together (like sphingolipids)

cholesterol

regulates membrane fluidity during normal cell growth and restricts random movement of phospholipid head groups at the outer surfaces of the leaflets

• it’s steroid ring interaction with the long hydrophobic tails of phospholipids immobilizes lipids and decreases biomembrane fluidity

decreased

In general, membrane fluidity is ___________ by sphingolipids and cholesterol and increased by phosphoglycerides.

membrane thickness

PC < PC + cholesterol < SM < SM + cholesterol

pure sphingomyelin (SM) bilayer

thicker than phosphoglycerides (phosphatidylcholine) bilayer

cholesterol lipid-ordering effect increases phosphoglyceride bilayer thickness

• lipid rafts are thicker than other membrane regions

membrane curvature

PC cylindrical shape - forms essentially flat monolayers

PE conical shape (smaller head group) - forms curved monolayers

lipid rafts

microdomains containing cholesterol, sphingolipids, and certain membrane proteins that form in the plane of the bilayer; these lipid-protein aggregates regulate signaling by certain plasma membrane receptors

• thicker than most bilayers

• enriched in glycoplipids

lipid droplets

storage compartments for triglycerides and cholesterol esters, also may serve as platforms for storage of proteins targeted for degradation

• formation:

  • cholesterol esters and triglycerides accumulate within the hydrophobic core of the lipid bilayer

  • delamination of the two lipid monolayers forms a “lens”

  • lens growth creates a spherical droplet released by scission at the neck

  • the newly formed droplet is surrounded by a lipid monolayer derived from the cytosolic leaflet of the ER membrane

saturated fats

type of fat containing high proportion of fatty acid molecules WITHOUT double bonds, considered to be less healthy in the diet

• ex: butter

unsaturated fats

healthy dietary fats characterized by having one or more double bonds in their fatty acid chains, making them liquid at room temperature

• ex: olive oil

trans fat

created as a side effect of partially catalytic hydrogenation of unsaturated plant fats (generally vegetable oils) with cis carbon-carbon double bonds

membrane-spanning domain

Integral membrane proteins contain one or more hydrophobic ___________

asymmetrically oriented

Transmembrane proteins and glycolipids are ___________ in the bilayer

asymmetry

typical single-pass transmembrane protein → glycophorin A

• glycosylation occurs solely on the exoplasmic side

dimeric glycophorin

• single (23-residue) membrane-spanning α helix

  • composed of amino acids with hydrophobic (uncharged) side chains

  • α helix typically 20-25 AA long in transmembrane proteins

• positively charged arginine and lysine residues near the cytosolic side of the helix bind negatively charged phospholipid head groups to anchor glycophorin in the membrane

• extracellular domain - heavily glycosylated; carbohydrate chains attached to specific serine, threonine, and asparagine residues

• cytosolic domain - interacts with cytoskeletal proteins

transmembrane domain

• hydrophobic side chains of the α helix interact with surrounding membrane lipids

• coiled-coil dimer interface; hydrophobic side chain van der Waals interactions between several AA

charged residues

polar or __________ in α-helical transmembrane segments can guide assembly an d stabilization of multimeric membrane proteins

T cell receptor (TCR) for antigen

composed of 4 separate dimers:

• αβ pair directly responsible for antigen recognition

• CD3 complex accessory subunits – γ, δ, ε, and ζ subunits

• Electrostatic attraction of positive and negative charges on each transmembrane domain forms the complete complex.

porins

pore-forming proteins that span the bilayer as a β-barrel

• barrel shaped unit

  • alternating outward-facing hydrophobic side chains on each β strand position the protein in the bilayer.

  • alternating inward-facing hydrophilic side chains line the pore.

  • β strands form the wall around a water-filled transmembrane pore in the center, through which small hydrophilic can diffuse.

• 16 antiparallel β-sheets

• hydrophobic side chains exposed to the bilayer

• hydrophobic residues exposed to pore

anchoring

covalently attached lipids anchor some otherwise water-soluble proteins to one or the other plasma membrane leaflet in eukaryotic cells

acylation

cytosolic proteins (such as v-Src) anchored to the PM through a single fatty acyl chain attached to N-terminal Gly

• common acyl anchors → myristate (C14) and palmitate (C16)

• v-Src: a viral mutant form of cellular tyrosine kinase, induces abnormal cellular growth that can lead to cancer when anchored to the membrane by myristylation

prenylation

cytosolic proteins (such as Ras and Rab G-proteins) anchored to the membrane through prenyl group thioether bond to one or two C-terminus Cys-SH groups

• CAAX box Cys prenylated and AAX removed)

• common anchors → unsaturated farensyl (C15) and greanylgeranyl (C20) groups

GPI (glycosylphosphatidylinositol) lipid anchor

anchors extracellular protein to exoplasmic surface of the PM

• phosphatidylinositol anchor → contains 2 fatty acyl chains inserted into bilayer

• phosphoethanolamine unit → links protein to the anchor

• sugar units → vary in number, nature, and arrangement in different anchors

• can cluster in lipid rafts

ABO blood types

• 3 structurally related oligosaccharides components of certain glycoproteins and glycolipids on the surface of human RBC and other cells

• the terminal oligosaccharide sugars distinguish the O, A, B, and AB antigens

• presence or absence of glycotransferases that add galactose (Gal), N-acetylgalactosamine (GalNAc), or both to the O antigen determines a person’s blood type

phospholipase A₂

structure - lipid-binding rim of positively charged arginine and lysine residues surrounds the catalytic active site cavity

catalysis - positively charged binding site rim residues bind to negatively charged polar groups at membrane surface

• small conformational change opens a channel to catalytic site lined with hydrophobic AAs

• phospholipid moves from the membrane leaflet into the channel

• enzyme-bound Ca ion binds the lipid head group, positions ester bond to be cleaved int he catalytic site

phospholipase

enzyme activity - each type hydrolyzes a specific bond in a phospholipid

functions:

• degrade damaged/aged cellular membranes

• generate signaling molecules

• contribute to destruction caused by many snake venoms

critical micelle concentration (CMC)

detergent concentration at which micelles (soapy bubbles) form

• concentration higher than CMC → detergent solubilizes lipids and integral membrane proteins, forming mixed micelles containing detergent, protein, and lipid molecules

• concentrations lower than CMC → non-ionic detergents dissolve membrane proteins without forming micelles by coating the protein membrane-spanning regions

ER

Fatty acids are synthesized in the _____ and moved to other membranes by multiple mechanisms

flippases

Phospholipids are asymmetrically distributed in the bilayer due to the action of _________

HMG-CoA reductase

__________ catalyzes the cholesterol biosynthesis rate-controlling step

fatty acid-binding protein (FABP)

small cytosolic proteins facilitate movement of fatty acids

• contain a hydrophobic pocket lined by β sheets that binds fatty acids

adipocyte FABP

has 2 β sheets, at right angles to each other, forming a clam shell structure

fatty acid interacts noncovalently with hydrophobic AA residues within this pocket

phospholipid synthesis in ER membrane

Step 1)

• 2 fatty acids synthesized on fatty acyl CoA - esterified to the phosphorylated glycerol backbone, forming a phosphatidic acid

• hydrocarbon tails anchor the molecule to the membrane

Step 2) phosphatase → converts phosphatidic acid into diacylglycerol (DAG)

Step 3) phosphotransferase transfers polar head group (ex: phosphorylcholine) from CDP-choline to the exposed OH group to make phosphatidylcholine

Step 4) flippase → uses ATP to catalyze movement of phospholipids from cytosolic leaflet to the exoplasmic leaflet to equalize leaflet growth and establish asymmetry

cholesterol biosynthetic pathway

1) HMG-CoA reductase (RATE CONTROLLING STEP)

• converts β-hydroxy-β-methylglutaryl CoA (HMG-CoA) to mevalonate

2) mevalonate → converted into IPP

3) IPP → converted into cholesterol and other lipids, through polyisoprenoid intermediate

cholesterol regulation

when there is high cholesterol levels in the ER membrane

• cholesterol binds to HMG-CoA reductase sterol-sensing domain

• causes interaction with integral ER membrane proteins (Insig-1 and Insig-2) which induces ubiquitnylation and degradation of HMG-CoA reductase by a proteasome

• reduces production of mevalonate, the key intermediate in cholesterol biosynthesis