Plasma Membrane I: Vacuoles, Proteasomes, and Transmembrane Transport

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Plasma Membrane IVacuoles, Proteasomes, and
Transmembrane Transport

• 22.228
• Dr. Bill Diehl-Jones

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Agenda

• Plant vacuoles
• Proteasomes
• Membranes
• Types
• Structure
• Transport methods
• Active
• Passive
• Facilitated

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Plant vacuoles.
Karp, fig. 4.8.36
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Plant Cell Vacuoles

• Plants lack lysosomes
• but vacuoles are analagous
• Bound by single membrane (tonoplast)
• Occupies up to 90 of cell volume
• Functions
• storage area for sugars, a.a., proteins,
  polysaccharides
• high solute concentrations maintain internal
  osmotic pressure, cell turgor
• works in concert with the cell wall to maintain
  plant structure

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Plant Cell Vacuoles contd

• end-point storage for metabolites,
• e.g. digitalis from foxglove plant
• toxic to herbivores and other enemies
• Like lipofuscin granules, can build up through
  life of the plant
• Homologous to lysosomes
• i. membrane bound H-ATPase
• ii. receives vesicles from TGN
• iii. permanent endpoint storage system

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Proteasomes

• Organelles which degrade proteins from the
  cytoplasm
• A protein structure
• not a lysosome, not a membrane-bound compartment
• Proteasomes are located within the cytoplasm and
  within the nucleoplasm
• Both prokaryotes and eukaryotes
• Direct uptake of proteins from cytoplasm/nucleopla
  sm
• Three related functions

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Functions of the Proteasome

• Housekeeping
• clearance of cytoplasm and nucleoplasm proteins
• each type of protein has a set half-life in the
  cytoplasm (steady turnover)
• metabolic enzymes, hemoglobin, structural
  proteins tend to last longer days to weeks
• regulatory molecules such as those that regulate
  DNA replication, are required only at certain
  times (S-phase) and have short life. their
  presence can be controlled by synthesis- N-end
  rule, protein half life correlates to the
  (single) N-terminal amino acid.
• if N-terminal is Arginine, lysine the protein
  lasts 2 - 3 min in cytoplasm
• if It is Val, Met, gt30 hrs

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Functions of the Proteasome

• Removal of improperly folded proteins
• mis-folded proteins are ejected from the R.E.R.
  back to cytoplasm via the translocon pores, then
  degraded by the proteasomes.

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Functions of the Proteasome

• Removal of ubiquitin - tagged proteins.
• a regulated step
• allows cell to remove certain proteins as needed
  (e.g. cyclin)
• Ubiquitin is a small, highly conserved protein
• Ubiquitin ligase, a family of related proteins
  recognize proteins destined for destruction, adds
  ubiquitin units
• Single ubiquitin on cytoplasmic face of
  trans-membrane proteins directs proteins to
  endosomes
• Multiple ubiquitin chains direct cytosol proteins
  to proteasomes

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Proteasomes StructureA barrel-like unit made of
globular proteins
Four rings, seven subunits each
cap protein
b-subunits (proteolytic function)
a-subunits
cap

• Together, these create a 13 angstrom opening
• Proteins must be unfolded and threaded in to
  the interior)

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Takin Out the GarbageThe ubiquitin tag

• 1. Protein tagged by a chain of ubiquitin
  molecules
• 2. Protein-ubiquitin complex binds to the cap
  protein
• 3. Ubiquitin removed, protein unfolded through
  cap and the a-subunit gap
• 4. b-subunits degrade protein to short peptides,
  return to cytoplasm

(nucleoplasm or cytosol)
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• Membranes An Overview
• Continuous, unbroken sheets, enclosing
  compartments
• Dynamic structures capable of fusing without
  losing continuity
• External membrane
• plasma membrane encloses contents of entire
  cells
• Internal membranes
• nuclear envelope
• mitochondrial membranes
• chloroplast membranes
• lysosomal membrane
• endoplasmic reticulum

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Summary of Membrane Functions
1. Compartmentalization 2. Provide a selectively
permeable membrane 3. Transporting solutes 4.
Responding to external signals (signal
transduction) 5. Intercellular interaction 6.
Locus for biochemical activities 7. Energy
transduction
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Overview of Plasma Membrane Structure a.
Biochemistry i. consists of polar lipids
arranged in a bilayer. ii. proteins iii.
carbohydrates b. Appearance of plasma membrane
in electron microscope i. thin sectioning
technique 7.5 nm wide with dark, light,
dark appearance ii. freeze fracture
technique - smooth areas interrupted by bumps
and depressions
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Fluid mosaic model a. lipid bilayer is core of
membrane b. lipid molecules are present in a
fluid state capable of rotating and moving
laterally within membrane. c. proteins occur as
mosaic of discontinuous particles. d. some
proteins penetrate deeply into, and even through,
lipid bilayer. e. membranes are dynamic
structures in which components are mobile. f.
components come together to engage in various
transient interactions.
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• Membrane lipids
• All are amphipathic
• except for glycolipids and cholesterol, all
  contain phosphate group
• these are phospholipids
• phospholipids have a glycerol backbone
• therefore, phospholipids are phosphoglycerides
• Four Types of lipids in cell membrane
• Phosphoglycerides
• Sphingolipids
• Glycolipids
• Cholesterol

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1. Phosphoglycerides - diglycerides - often
one saturated and one unsaturated fatty acid. -
3rd OH group has phosphate plus either 1.
choline - phosphatidyl choline. 2. ethanolamine
- phosphatidylethanolamine. 3. serine -
phosphatidylserine. 4. inositol -
phosphatidylinositol.
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• 2. Sphingolipids
• - sphingosine-based lipids.
• - sphingosine is an amino alcohol with a long
  hydrocarbon chain.
• two additions
• a. always a fatty acid to amino group of
  sphingosine
• This is a ceramide.
• b. additional groups esterified to terminal OH.
• i. if phosphorylcholine, molecule is
• sphingomyelin.
• ii. if carbohydrate, molecule is glycolipid.

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3. Glycolipids - carbohydrate attached to
sphingolipid (ceramide) - if carbohydrate is a
monosaccharide, glycolipid is a cerebroside. -
if carbohydrate is an oligosaccharide, glycolipid
is a ganglioside. 4. Cholesterol (see Fig.
4.7, Karp, p. 130) - may constitute up to 50 of
lipid molecules in plasma membrane of certain
animal cells. - is absent from plasma membranes
of all bacterial cells, and most plant cells. -
cholesterol increases fluidity of bilayer.
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Membrane Carbohydrates - all of carbohydrate
faces outward into extracellular space. -
covalently linked to either protein or lipid. 1.
Glycoproteins - carbohydrate is present in
short, branched oligosaccharides (lt15 sugars per
chain) - may be attached to R group of 1.
asparagine (N-linked) 2. serine or threonine
(O-linked) - play roles in cell interactions. 2.
Glycolipids - may play role in certain
infectious diseases. - carbohydrates of
glycolipids determine blood type (A, B, AB or O)
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Membrane proteins - 12 to gt50 different
ones. - asymmetrically situated. - properties
of outer portion of membrane are very different
from properties of inner portion. - 3 distinct
classes, based on their relationships to lipid
bilayer
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1. Integral membrane proteins - penetrate into
lipid bilayer. - usually pass right
through. - have segments protruding into
extracellular space. - have segments
protruding into cytoplasm. - transmembrane
segments - pass through lipid bilayer. -
usually consist of nonpolar amino acids
organized in an ?-helical conformation. - some
integral membrane proteins can form aqueous
channel through lipid bilayer.
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2. Peripheral membrane proteins - located
entirely outside of lipid bilayer. - either on
extracellular or cytoplasmic surface. -
associated with membrane surface by noncovalent
bonds. - weak electrostatic bonds to hydrophilic
head groups of phospholipids or hydrophilic
portion of integral proteins. - peripheral
proteins on cytoplasmic surface function in
transmembrane signal transduction.
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3. Lipid-anchored membrane proteins -
covalently linked to lipid molecules within
bilayer. - two types of lipid anchors a.
glycophosphatidyl inositol in outer
leaflet proteins linked to this by short
oligosaccharide chain b. long
hydrocarbon chains in inner leaflet A G
protein called Ras is linked this way. G
proteins are involved in transmembrane signal
transduction.
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Movement within Membranes 1. Control over
membrane motility a. integral membrane proteins
are not totally free to drift in lipid sea. b.
restraints as a result of i. interactions
occurring within membrane. ii. links to
materials on inner or outer surface of
membranes. (see Fig. 4.28, Karp, p.
146) c. leads to membrane domains -- regions in
which a protein may travel (see Fig. 4.29, Karp,
p. 147)
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Movement Within the Cell Membrane
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2. Membrane Domains and Cell polarity e.g.
epithelial cells lining inner surface of
intestine. - highly polarized cells whose
different surfaces carry out different
functions. a. apical plasma membrane -
faces lumen of intestine - selectively
absorbs substances from lumen. b. lateral plasma
membrane surface - interacts with
neighbouring epithelial cells. c. basal plasma
membrane - adheres to underlying
extracellular substrate (basement membrane)
- functions in exchange of substances with
bloodstream.
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Enterocytes In Situ
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Differentiated Enterocyte
Glycocalyx
Occluding Junction
Tight Junction
Gap Junction
Desmosome
Basement Membrane
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Factors Affecting Enterocyte Barrier Integrity
Transport
Cytoskeleton
Differentiation
Apoptosis
Pro- and Anti-inflammatory Mediators
Junctional Coupling
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A Neat Trick

• Measuring Confluency of Monolayers

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Transepithelial Electrical Resistance

• A method for determining confluency of CaCo-2
  cultures
• Resistance is measured across the
  apical/baso-lateral surface
• A DECREASE in resistance indicates loss of
  barrier function

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Membrane Transport I. Permeability of Plasma
Membranes 1. Movement of substances across
plasma membranes - plasma membrane is
selectively permeable. a. i.e. some solutes
are more permeable than others. b. this
allows cells to concentrate subtances c.
influx - movement of substance into cells.
d. efflux - movement of substances out of
cells. e. net flux - one exceeds the
other. - plasma membrane is semipermeable.
a. i.e. freely permeable to H2O (solvent), while
allowing much slower passage to small ions and
polar solutes.
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II. Diffusion and Osmosis 1. Diffusion -
continual movement of molecules among each other
in liquids or gases - greater the concentration
difference the greater is the rate of diffusion.
(cells can manipulate this in potocytosis) -
ability of an ion to diffuse between two
compartments depends on 2 gradients 1.
chemical gradient determined by concentration
difference of substance. 2. electric
potential gradient determined by difference in
charge. - together these differences are
combined to form an electrochemical gradient.
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2. Diffusion of Water through Membranes Osmosis
the movement of water through a semipermeable
membrane in response to a concentration gradient
of H2O. - Osmotic pressure is proportional to
the number of particles in solution -
Osmolarity is a calculated quantity of a
solution. - Units are milliosmoles.
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Tonicity - an observed property that depends on
permeability of the cell membrane and is relative
to another cell or solution (Figs. 4.33, 4.34,
Karp, p. 153). a. If cell swells, the solution
is hypotonic relative to cell. i. animal cells
swell and eventually burst. - If these are red
blood cells, this is hemolysis. ii. plant cells
push against surrounding cell wall. - This is
turgor pressure. (Fig. 4.35 Karp, p. 150) b. If
cell shrinks, the solution is hypertonic relative
to cell. i. Plant cell pulls away from
surrounding cell wall. - This is plasmolysis.
c. If cell neither shrinks nor swells, the
solution is isotonic.
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Osmolarity and Tonicity(Are they different?)
Isoosmotic Soln

• You bet!
• Osmolarity
• R/t no. dissolved solutes
• Tonicity
• R/t effect of s solute on cell membrane

Hyperosmotic Soln
Hypoosmotic Soln
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Tonicity
Isotonic Soln

• Does it shrink or swell or stay the same?
• When might an isoosmotic solution not be
  isotonic?

Hypertonic Soln
Hypotonic Soln
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Plant Cell in Hypertonic Environment
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Free Energy and Solute Movement Across
Membranes For a solute moving into a cell
Wherez charge of solute F a constant ?Em
potential difference across membrane If
solutein/soluteout lt 1, then log(in/out) is
negative. If solutein/soluteout gt 1, then
log(in/out) is positive. If solutein/soluteout
1, then log(in/out) 0. If z 0, then
zF?Em 0. If z and ?Em have the same sign,
zF?Em is positive. If z and ?Em have different
signs, zF?Em is negative.
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How do we measure Em?Note that the membrane
potential is negative
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K
K
K
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K
K
K
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Solute Transport Mechanisms
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1. Overview a. Passive - diffusion - all
driven by concentration gradient (electrochemical
gradient in the case of charged solutes). - all
passive transport is thermodynamically
spontaneous i. simple diffusion - through lipid
bilayer ii. simple diffusion - through an
aqueous channel iii. facilitated diffusion -
solute binds specifically to a membrane protein
carrier. b. Active - energy coupled
transport - all active transport is
non-spontaneous (requires input of free
energy) - uses protein carrier but driven by
ATP hydrolysis.
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2. Diffusion through lipid bilayer a(i)
above 1. Greater the lipid solubility, faster
the diffusion into the cell. (see Fig. 4.32,
Karp, p 152) - Partition coefficient is a
measure of lipid solubility or hydrophobicity or
nonpolarity. - partition coefficient
concentration in oil concentration in
H2O 2. The smaller the molecule, the greater is
the rate of diffusion. - Very small, uncharged
molecules penetrate very rapidly. e.g. O2 , H2O,
NO CO2 appear to slip between adjacent
phospholipids 3. ions and larger polar
molecules, such as sugars amino acids, cannot
diffuse through lipid bilayer.
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3. Diffusion of ions through membranes a(ii)
above ion channels - permeable to specific
ions. - each formed by integral membrane
proteins that surround an aqueous pore. - highly
selective - only one type of ion passes through
pore. - bidirectional. - net flux depends on
electrochemical gradient.
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Gated channels a. Most ion channels exist in
either an open or a closed conformation b. Such
channels are termed gated. c. Voltage-gated
channels - conformation state depends on
difference in ionic charge on two sides of
membrane. e.g. K channel d. Chemical-gated
channels - conformational state depends
upon binding of a particular substance. e.g.
acetylcholine (neurotransmitter) acts on outer
surface of certain cation channels.
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V-Gated Na ChannelsAltered States
Open
Inactivated
Closed
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Voltage-gated K channel
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4. Facilitated diffusion a(iii) above -
selective binding of substance to a
membrane-spanning protein, a facilitative
transporter, improves permeability of a substance
without altering direction in which substance
would tend to move on its own. - does not
require ATP. - movement facilitated equally well
in both directions. - direction depends on
concentration gradient.
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Facilitated diffusion mechanism 1. solute binds
to facilitative transporter on one side of
membrane. 2. triggers a conformation change in
protein. 3. solute is exposed to other surface
of membrane. 4. solute can then diffuse down its
concentration gradient.
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Certain similarities between facilitative
transporters enzymes... 1. both specific for
molecules they transport. 2. both have their
activities subject to regulation. 3. both
exhibit saturation-type kinetics.
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5. Active transport - selective binding of
substance to a membrane-spanning protein, an
active transporter, allows passage of substance
through membrane against electrochemical gradient
for that substance - active transporter acts by
undergoing conformational change upon binding
substance. - substance is moved against a
concentration gradient. - movement occurs in
only 1 direction. - requires ATP or other form
of energy such as flow of other substances down a
gradient ex Na-K ATPase or sodium-potassium
pump
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Na-K ATPase - 3 Na pumped out to 2 K pumped
in for each ATP consumed. - electrogenic
pump. - contributes directly to separation of
charge across membrane. - Na gradient provides
a means by which free energy can be stored in
cell. - this potential energy in ionic gradient
can be utilized in various ways.
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Na-K ATPase transport cycle 1. Na ions bind
to protein on inside of membrane. 2. ATP is
hydrolyzed. 3. conformation of Na-K ATPase is
changed. 4. Na ions are expelled to external
space. 5. K ions bind on outside of
membrane. 6. phosphate group on protein is
removed. 7. protein snaps back to its original
conformation. 8. K ions move to inside of cell.
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Coupling active transport to existing ion
gradients 1. Na that has been pumped out is
driven back in by concentration gradient. 2.
Membrane almost impermeable to Na. 3. Therefore
transport protein is required. 4. Transport
protein does not work unless it binds another
molecule (e.g. glucose). 5. Glucose is driven
into cell against its concentration gradient. 6.
This is cotransport and driven by secondary
active transport. 7. Symport - two solutes are
moved in same direction. (i.e. glucose
and Na) 8. Antiport - two solutes moved in
opposite direction. (i.e. Na and H)
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Secondary transport in intestine (see Figs. 4.29
and 4.43, Karp, p. 147 and 165) 1. uptake of
glucose by cotransport occurs at apical plasma
membrane. 2. glucose diffuses through cytoplasm
of intestinal epithelial cell. 3. at basal
plasma membrane glucose is transported out of
cell and into bloodstream by facilitated
diffusion.
Karp Fig 4.43 Secondary Transport
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IV. Membrane potentials - all cells have a
voltage gradient (charge gradient) across their
membrane (membrane potential). - by
convention, inside is negative with respect to
outside. - magnitude varies between -15 and
-100 mV. - membrane potential arises from
1. passive properties of the membrane
(semi-permeable) 2. active transport in
Na/K ATPase