Medical BiochemistryMedical Biochemistry

Membranes: Bilayer Properties, Transport

Lecture 72

Membranes: Bilayer Properties, Transport

Lecture 72

Synthesis of secretory proteins - review1. N-terminal signal sequence is synthesized

2. Signal bound by SRP, complex docks with SRP receptor on ER membrane

3. Signal sequence binds to translocon, internal channel opens, inserted into translocon

4. Polypeptide elongates, signal sequence cleaved

5. ER chaperones prevent faulty folding, carbohydrates added to specific residues

6. Ribosomes released, recycle

7. C-terminus of protein drawn into ER lumen, translocon gate shuts, protein assumes final conformation

Synthesis of integral membrane protein

• Integral membrane protein may, or may not have N-terminal signal sequence

• In absence of N-terminal signal sequence, internal signal sequence bound by SRP

•Animation:ERimport.mov

• SRP-protein-ribosome complex docks with SRP receptor, C-terminal portion of protein cotranslationally inserted into lumen of ER

•Mature protein transverses ER bilayer forming integral membrane protein

•NOTE: Orientation of protein within membrane dependent upon cluster of charged residues adjacent to internal signal sequence

•In presence of N-terminal signal sequence, integral membrane protein produced by stop-transfer signal that forms transmembrane domain

Secretory Pathway

• Once a protein has entered exocytotic pathway, in general, it never returns to cytosol (notable exception is misfolded proteins - retrograde transport for degradation)

• In the absence of a sorting signal, protein will follow constitutive secretory pathway (i.e., directed to plasma membrane) in transport vesicles

• Some proteins contain retention signals (e.g., KDEL in C-terminus of some ER proteins)

Secretory Pathway

• In specialized cells, regulated secretory pathway leads to packaging of product in secretory vesicles

Asymmetry of proteins and lipids maintained during membrane assembly

• Orientation of a protein (asymmetry) is determined upon entry into ER, does not change during transit to other membrane/organelle

• Fusion of a vesicle with the plasma membrane preserves the orientation of any integral proteins embedded in the vesicle bilayer

• Animation: Secretion.mov

Small GTPases Act as Molecular Switches

GDP GTP

GTP GDP

“Inactive” “Active”GEF

Pi

GAP

GTP exchange for bound GDP, facilitated by Guanine-nucleotide Exchange Factors (GEFs), “activates” protein (usually resulting in conformational change). Hydrolysis of GTP GDP, accelerated by GTPase-Activating Proteins (GAPs), “inactivates” complex.

ARF - vesicular transport

Ran - nuclear transport

Rab - regulated secretion, endocytosis, intracellular transport

Rho - formation of actin cytoskeleton

Ras - growth and differentiation signaling pathways

Intracellular Transport Vesicles

Step 1: Coat assembly initiated

Step 2: ARF recruits coat proteins

Step 3: Vesicle budding

Step 4: Coat disassembly

Step 5: Vesicle targeting (v-SNARE)

Step 6: General fusion machinery assembles (NSF, SNAP)

Step 7: Vesicle fusion

Step 8: Retrograde transport

NOTE: Botulinum B toxin, one of most lethal toxins known (most serious cause of food poisoning), is a protease that cleaves synaptobrevin (one v-SNARE involved in fusion of synaptic vesicles) and inhibits release of acetylcholine at neuromuscular junction. Possibly fatal, depending on dose taken.

Signal sequences target proteins to their correct destinations

• Signal sequences identified for cytosolic proteins destined for nucleus, mitochondria, peroxisomes

• Animation: Targeting.mov

• Nuclear import via nuclear pore complex. Bidirectional transport, accomodates large, complex structures (e.g., ribosomes), nuclear localization signal (NLS) not cleaved during transport.• Mitochondrial (mt) genome encodes 13 proteins, must import remainder. Matrix proteins must pass through outer and inner mt membranes. Proteins must be unfolded by chaperone proteins before translocation. Signal sequence usually cleaved.

• Peroxisomes can import intact oligomers (e.g., tetrameric catalase). Zellweger Syndrome - mutation in genes (peroxins) involved in peroxisome biogenesis (or certain peroxisomal enzymes)

Major mechanisms used to transfer material and information across membranes

Cross-membrane movement of small moleculesDiffusion (passive and facilitated)Active Transport

Cross-membrane movement of large moleculesEndocytosisExocytosis

Signal transmission across membranesCell surface receptors

1. Signal transduction (e.g., glucagon cAMP)2. Signal internalization (coupled with endocytosis, e.g., LDL receptor)

Movement to intracellular receptors (steroid hormones; a form of diffusion)

Intercellular contact and communication

Table 43-11

Passive Mechanisms Move Some Small Molecules Across Membranes

• Passive transport down electrochemical gradients by simple or facilitated diffusion– passive diffusion (e.g., gases) limited by concentration

gradient across membrane, solubility of solute, thermal agitation of that specific molecule

• Active transport, against gradient, requires energy

Ion Channels Selectively Transport Charged Molecules

• Specific channels for Na+, K+, Ca2+, and Cl- have been identified

• Channels are very selective, in most cases, to only one type of ion

• Subset of K+ channels (“K+ leak channels”) open in “resting” cell– make plasma membrane more permeable to K+ than

other ions, maintains membrane potential

Activities of Ion Channels Can Be Regulated

• Channels are “gated” - open transiently– Ligand-gated channels - specific molecule binds receptor, open

channel (e.g., acetylcholine)

– Voltage-gated channels - open (or close) in response to changes in membrane potential

• Ion channel activities are affected by certain drugs

• Mutations in genes encoding ion channels can cause specific diseases (e.g., Cystic fibrosis - mutations in CFTR, a Cl- channel)

Net diffusion of substance depends on:

• Its concentration gradient across membrane - solutes move from high to low concentration

• Electrical potential across membrane - solutes move toward solution with opposite charge (inside of cell usually has negative charge)

• Permeability coefficient of substance

• Hydrostatic pressure gradient across membrane - pressure will rate and force of collision with membrane

• Temperature - temperature will particle motion and frequency of collisions between particles and membrane

Types of transport systems

• Classified by direction of movement and whether one or more unique molecules are moved– Uniport system moves one type of molecule

bidirectionally– Cotransport systems transfer one solute dependent upon

simultaneous or sequential transfer of another solute• Symport - moves solutes in same direction (e.g., Na+-sugar

transporters or Na+-amino acid transporters)• Antiport - moves two molecules in opposite directions (e.g.,

Na+ in and Ca2+ out)

Transport with carrier proteins

• Facilitated diffusion and active transport used to transport molecules that cannot pass freely through lipid bilayer by themselves– Both involve carrier

proteins; show specificity for ions, sugars, and amino acids; and resemble a substrate-enzyme reaction (but with no covalent interaction)

– But, facilitated diffusion can be bidirectional, while active transport usually unidirectional

– And, active transport always against gradient, requires energy

– Specific binding site for solute

– Carrier is saturable (has maximum rate of transport - Vmax)

– There is a binding constant (Km) for the solute, so the whole system has a Km

– Structurally similar competitive inhibitors block transport

Facilitated Diffusion

• Some solutes diffuse across membranes down electrochemical gradients more rapidly than expected from size, charge, and partition coefficients

• “Ping-Pong” mechanism explains facilitated diffusion

• Carrier protein exists in two principal conformations:– “Pong” state - exposed to high [solute], solutes bind to specific sites on

carrier protein– Conformational change exposes carrier to lower [solute] - “ping” state– Process is reversible, net flux depends on concentration gradient

Facilitated Diffusion

• Rate of solute entry into cell determined by:– Concentration gradient across the membrane– Amount of carrier available (key control step)– Rapidity of solute-carrier interaction– Rapidity of conformational change (both loaded and

unloaded carrier)

• Hormones regulate by changing number of transporters available– e.g., insulin increase glucose

transport in fat and muscle by recruiting transporters from intracellular reserve

Active Transport• Transport away from thermodynamic equilibrium

– Energy is required (from hydrolysis of ATP, electron movement, or light)

– Maintenance of electrochemical gradients in biologic systems consumes ~30-40% of total energy expenditure of cell

• Cells, in general, maintain low intracellular [Na+] and high intracellular [K+], with net negative electrical potential inside– Gradients maintained by Na+-K+ ATPase– Ouabain or digitalis (cardiac glycosides used to

treat congestive heart failure) inhibits ATPase by binding to extracellular domain. (Raises intracellular [Na+], Na+/Ca2+ antiporter functions less efficiently with lower [Na+] gradient, thus fewer Ca2+ ions exported, intracellular [Ca2+] increases causing muscle to contract more strongly.)

Glucose Transport - Several Mechanisms

• In adipocytes and muscle, glucose enters by facilitated diffusion

• In intestinal cells, glucose and Na+ bind to different sites on glucose transporter (symport)– Na+ enters cell down electrochemical

gradient and “drags” glucose with it

– To maintain steep Na+ gradient, Na+-glucose symport depends on low intracellular [Na+] maintained by Na+-K+ pump

– A uniport allows glucose accumulated in cell to move across different membrane toward a new equilibrium

Endocytosis

• Process by which cells take up large molecules– Source of nutritional elements (e.g., proteins,

polynucleotides)

– Mechanism for regulating content of certain membrane components (e.g., hormone receptors)

– Most endocytotic vesicles fuse with lysosomes• hydrolytic enzymes digest macromolecules (yields amino acids,

simple sugars, and nucleotides)

– Two general types of endocytosis• Phagocytosis - specialized cells (e.g., macrophages) ingest large

particles (viruses, bacteria)

Endocytosis

• Pinocytosis - property of all cells– Fluid-phase pinocytosis - nonselective uptake of a solute by small

vesicles• loss of membrane replaced by exocytosis

– Absorptive pinocytosis - receptor-mediated selective process

• permits selective concentration of ligands from medium, limits uptake of fluid or soluble unbound macromolecules

• vesicles derived from coated pits (clathrin)

• fate of receptor/ligand depends of particular receptor– e.g., LDL receptor recycled, LDL processed in lysosomes

– EGF receptor degraded (receptor downregulation)

Fluid-phase Receptor-mediatedendocytosis endocytosis

Exocytosis

• Most cells release macromolecules to the exterior

• Signal for regulated exocytosis is often a hormone– binds to cell-surface receptor, induces local and transient

change in [Ca2+] that triggers exocytosis

• Molecules released by exocytosis fall into 3 categories– Attach to cell surface and become peripheral proteins (e.g.,

antigens)

– Become part of extracellular matrix (e.g., collagen)

– Enter extracellular fluid and signal other cells (e.g., insulin)

Mutations Affecting Membrane Proteins Cause Diseases

• Membrane proteins classified as: receptors, transporters, ion channels, enzymes, and structural components

• Member of each class often glycosylated– mutations affecting this process may alter function

Disease Gene Mutation Protein type

Achondroplasia Fibroblast growth factor receptor 3 Receptor

Familialhypercholesterolemia

LDL receptor Receptor

Cystic fibrosis CFTR protein Cl- transporter

Congenital long QTsyndrome

ion channels in heart ion channels

Wilson disease copper-dependent ATPase enzyme