Medical Biochemistry

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Medical Biochemistry

• Membranes Bilayer Properties, Transport
• Lecture 72

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Synthesis of secretory proteins - review
1. 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
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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
• AnimationERimport.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

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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)

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Secretory Pathway

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

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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

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Small GTPases Act as Molecular Switches
ARF - vesicular transport Ran - nuclear
transport Rab - regulated secretion, endocytosis,
intracellular transport Rho - formation of actin
cytoskeleton Ras - growth and differentiation
signaling pathways
GEF
Inactive
Active
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.
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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.
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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)

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Major mechanisms used to transfer material and
information across membranes
Cross-membrane movement of small
molecules Diffusion (passive and
facilitated) Active Transport Cross-membrane
movement of large molecules Endocytosis Exocytosis
Signal transmission across membranes Cell
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) Intercellu
lar contact and communication
Table 43-11
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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

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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

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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)

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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

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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)

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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

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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

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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

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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.)

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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

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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)

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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)

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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)

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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