Transcript Chapter 8-1

Chapter 5:
Aerobic Respiration
and the Mitochondrion
Mitochondrial outer membrane
• ~50% lipid by weight
• Contains many enzymes involved in diverse
activities: epinephrine oxidation, tryptophan
degradation, fatty acid elongation, etc.
• Porin channel is surrounded by a barrel of β
strands
• If porin channels wide open, outer
membrane is freely permeable to molecules
like ATP, NAD & coenzyme A
Porins
• Molecules up to ~5,000 daltons to penetrate
• The intermembrane space & cytoplasm are
basically continuous with respect to ATP,
NAD, CoA, etc.
Mitochondrial inner membrane
• Very high protein/lipid ratio
(3:1 by weight; ~1 protein/every 15 phospholipids)
• >100 different polypeptides; devoid of cholesterol
• Rich in the unusual phospholipid cardiolipin
• Both the presence of cardiolipin & the absence of
cholesterol are characteristic of bacterial plasma
membranes
Mitochondrial inner membrane
• Ca2+-ATPase
• Electron transport chain
• ATP synthase
Mitochondrial matrix
• Enzymes
• Ribosomes
• Circular double-stranded DNAs (encode inner
membrane proteins; nuclear DNA codes for some,
too)
• Humans mitochondrial DNA encodes
– 13 mitochondrial polypeptides
– rRNAs and 22 tRNAs that are used in protein
synthesis within the organelle
The role of anaerobic and aerobic
metabolism in exercise
• Muscle cells contain a store of creatine
phosphate (CrP )
• CrP + ADP Cr + ATP
• Human skeletal muscles consist of fasttwitch fibers and slow-twitch fibers
Fast-twitch fibers
• Contract very rapidly; 15 – 40 msec
• Nearly devoid of mitochondria
• Unable to make much ATP by aerobic
respiration
Slow-twitch fibers
• Contract more slowly; 40 – 100 msec
• Have large numbers of mitochondria
Aerobic exercise
• Energy source
– Initially by glucose stored as glycogen in
muscles
– After a few minutes the muscles depend
increasingly on free fatty acids released into
blood from adipose (fat) tissue
• The longer the exercise period, the greater
the dependency on fatty acids
Direct evidence for rotation of γ subunit
relative to αβ subunits
• Prepared a genetically engineered version of
working part of ATP synthase (3α, 3β & a γ
[α3β3γ])
• Fixed polypeptide complex to glass coverslip by
its head & attached short, fluorescently labeled
actin filament to γ subunit end jutting into medium
• Add ATP & rotation seen (like propellor)
• Powered by energy released as ATPs were bound
& catalyzed by β subunit catalytic sites
The mechanism by which H+ movement
drives c ring rotation
• Each a subunit has 2 half-channels that are
physically separated (offset) from one another
• One half-channel leads from intermembrane
(cytosolic) space into the middle of the a subunit;
the other leads from the middle of the a subunit
into the matrix
• Each proton moves from the intermembrane space
through the half-channel & binds to a negatively
charged Asp residue situated at the surface of the
adjoining c subunit
The mechanism by which H+ movement
drives c ring rotation
• Binding of H+ to Asp carboxyl group generates a
major conformational change in the c subunit that
causes the subunit to rotate ~30° in a
counterclockwise direction
• This movement of the recently protonated c
subunit brings the adjoining ring subunit
(protonated at an earlier step) into alignment with
the second a subunit half-channel
The mechanism by which H+ movement
drives c ring rotation
• The Asp releases its associated proton,
which diffuses into the matrix
• After proton dissociation, the c subunit then
returns to its original conformation & is
ready to accept another proton from the
intermembrane space & repeat the cycle
Peroxisomes
• Found in 1954 & called microbody
• Simple membrane-bound vesicles with 0.1 - 1.0
µm diameter
• Often have dense, crystalline core of an oxidative
enzyme(s) & consequently granular appearance
• Multifunctional organelles containing >50
enzymes involved in diverse activities like:
– Oxidation of very long chain fatty acids
(VLCFAs); whose chains typically contain 24 –
26 C
Peroxisomes
• Synthesis of plasmalogens
– Abnormalities in plasmalogen synthesis can
lead to severe neurological dysfunction
• Luciferase
– which generates light emitted by fireflies, is
also a peroxisomal enzyme
Peroxisomes
• Named peroxisomes since they are the site of
synthesis & degradation of H2O2
• H2O2 is produced by a number of peroxisomal
enzymes
– Urate oxidase, glycolate oxidase & amino acid
oxidases that utilize molecular oxygen to
oxidize their respective substrates
• Catalase (at high concentration in peroxisomes)
rapidly breaks down H2O2 generated in these
reactions
Peroxisomes
• Form by splitting from preexisting
organelles
• Import preformed proteins from cytosol
• Do similar kinds of oxidative metabolism in
mitochondria
– Alanine/glyoxylate aminotransferase, is seen in
the mitochondria of some mammals (cats, dogs)
& peroxisomes of others (rabbits, humans)
Glyoxysomes
• A specialized type of peroxisome found
only in plants
• Contain some of same enzymes (catalase,
fatty acid oxidase), but others as well
• Plant seedlings rely on stored fatty acids to
provide energy & material to form new
plant
• Glyoxylate cycle
Glyoxysomes
• A primary metabolic activity in these
germinating seedlings is the conversion of
stored fatty acids to carbohydrate
• Stored fatty acid disassembly produces
acetyl CoA & it condenses with
oxaloacetate to form citrate
• Citrate is then converted to glucose by a
series of glyoxylate cycle enzymes found in
glyoxysomes
Diseases result from abnormal
mitochondrial or peroxisomal function
• Muscle & nerve tissues tend to be most seriously
impacted in these disorders since they have the
highest demand for ATP
• Depending on protein(s) affected, conditions vary
in severity from diseases that lead to death during
infancy to disorders that produce seizures (中風驟發),
blindness, deafness and/or strokelike episodes
• Sometimes conditions are mild & characterized by
intolerance to exercise or nonmotile sperm
Abnormal mitochondria
• Closer examination of mitochondria reveals large
numbers of abnormal inclusions
• A number of common neurological diseases with
adult onset (like Parkinson's disease) might be a
consequence of degenerative changes in
mitochondrial function
• The first such disease-causing mutation was
reported in 1995
– The mutation occurred in gene encoding the
flavoprotein subunit of the TCA enzyme
succinate dehydrogenase
Mitochondrial disorder inheritance
contrasts in several ways with nuclear
gene Mendelian inheritance
• Mitochondria in cells of human embryo are
derived exclusively from mitochondria present in
the egg at the time of conception without any
contribution from the fertilizing sperm
• Mitochondrial disorders are inherited maternally
• Mitochondria in cell can contain mixture of
normal (wild-type) & mutant mtDNA
(heteroplasmy)
mtDNA Mutation
• Nuclear DNA is protected from damage by
a variety of DNA repair systems which are
generally lacking in mitochondria
• mtDNA may also be subjected to high
levels of mutagenic oxygen radicals
• mtDNA experiences >10 times the mutation
rate of nuclear DNA
Abnormal peroxisomes
• Zellweger syndrome (ZS) is a rare inherited
disease characterized by a variety of neurological,
visual & liver abnormalities leading to death
during early infancy
• Sidney Goldfischer et al. (1973) – reported that
liver & renal cells from these patients lacked
peroxisomes
• Later studies showed that peroxisomes were not
entirely absent from the cells of these individuals
Zellweger syndrome (ZS)
• Peroxisomes were present as empty membranous
ghosts (organelles lacking the enzymes normally
found in peroxisomes)
• These individuals can make peroxisomal enzymes
but the enzymes fail to be imported into
peroxisomes & stay largely in cytosol where they
are unable to carry out their normal functions
• Mutations in at least 11 different genes
– Encoding proteins involved in uptake of
peroxisomal enzymes from cytosol
Adrenoleukodystrophy (ALD), subject
of the movie Lorenzo's Oil
• Absence of a single peroxisomal enzyme
• A defect in a membrane protein that transports
very-long-chain-fatty-acids (VLCFAs) into the
peroxisomes where they are normally metabolized
• In the absence of this protein, VLCFAs
accumulate in brain & destroy myelin sheaths that
insulate nerve cells
• Boys with the disease are typically unaffected
until midchildhood, when symptoms of adrenal
insufficiency & neurological dysfunction begin
Adrenoleukodystrophy (ALD)
• A diet rich in certain fatty acids is able to retard
the progress of the disease
• A number of ALD patients have been successfully
treated by bone marrow transplantation, which
provides normal cells capable of metabolizing
VLCFAs
• Administration of drugs (e.g., lovastatin) that may
lower VLCFA levels
• Clinical studies employing gene therapy are also
being planned
Chapter 8-1:
Cytoplasmic Membrane Systems:
Structure, Function, and
Membrane Trafficking
Endomembrane system
• Plasma membrane, vesicles, vacuoles, ER,
Golgi apparatus, nuclear membrane,
lysosome
– Have distinct structures & functions but
together form an endomembrane system
– Dynamic, integrated network
– Materials are shuttled (transport vesicles)
between the endomembrane system
Transport vesicles in endomembrane
system
• Transport vesicles form by budding from
donor compartment
• Transport vesicles move in directed manner,
often pulled by motor proteins operating on
tracks formed by microtubules &
microfilaments of the cytoskeleton
• When they reach their destination, they fuse
with acceptor compartment
Transport in endomembrane system
• Endocytic pathway
• Exocytotic pathway
– Secretory pathway
Biosynthetic (secretory) pathway
• Synthesis in ER (protein) or Golgi (lipid,
carbohydrate)
• Many materials made in ER (proteins) &
Golgi (complex polysaccharides) fated for
secretion from cell
• Two types of secretory activity
– Constitutive secretion
– Regulated secretion
Constitutive secretion
• Synthesis & secretion into extracellular
space occurs in continual, unregulated
manner
• Form extracellular matrix & plasma
membrane
Regulated secretion
• Secretory materials stored in large, densely
packed, membrane-bound secretory
granules in cell periphery
• Secreted after correct stimulus
– Endocrine cells release hormones
– Pancreatic acinar cells release digestive
enzymes
– Nerve cells release neurotransmitters
Proteins targeting
• Through sorting signals located on proteins &
receptors in transport vesicle walls that recognize
them
• Salivary gland cell protein trafficking
– Salivary mucus proteins (made in ER)
specifically targeted to secretory granules
• Lysosome enzymes (also made in ER) specifically
sent to lysosome
• Sorting signals are encoded in protein amino acid
sequence or in attached oligosaccharides
Approaches to the study of
cytomembranes
• EM micrographs give detailed view of cell
cytoplasm, but little insight into functions of
the structures
• Insights gained from autoradiography
– Detect location of radioactively labeled
materials in cell
• Insights from pulse-chase trials
Pulse-chase trials
• Expose to hot amino acids briefly (pulse)
• Wash to remove excess isotope from tissue
• Transferred tissue to medium with
unlabeled amino acids (chase), which lasts
for varying time periods
• See wave of radioactivity moving through
cell, discern pathway sequence
Use of green fluorescent protein (GFP)
reveals the movement of proteins within
a living cell
• GFP is small protein from certain jellyfish
that emits a green fluorescent light
• GFP gene fused to DNA encoding protein to
be studied
• Introduce the chimeric DNA into cells
• Chimeric DNA expresses chimeric protein
(GFP fused to the protein to be studied)
Use of green fluorescent protein (GFP)
reveals the movement of proteins within
a living cell
• Usually, GFP stuck to end of a protein has
little or no effect on its movement or
function & protein under study has no effect
on fluorescence of attached GFP
Example: infect a mammalian cell with vesicular
stomatitis virus (VSV) strain in which a viral gene
(VSVG) is fused to GFP gene
• Cell begins to make massive amounts of VSVG
protein in RER
• VSVG then goes to Golgi complex & eventually
to the plasma membrane of the infected cell where
they are incorporated into viral envelopes
• Can see relatively synchronous wave of protein
movement (green fluorescence) soon after
infection
Infect a mammalian cell with vesicular
stomatitis virus (VSV) strain in which a viral
gene (VSVG) is fused to GFP gene
• Synchrony is enhanced by use of virus with
mutant VSVG protein that cannot leave ER
of infected cells grown at elevated
temperature (40°C).
• The green fluorescence is restricted to the
ER.
• When temperature is lowered to 32°C, the
fluorescent GFP-VSVG protein that had
accumulated in ER moves synchronously to Golgi
complex for various processing events & then to
membrane
• Temperature-sensitive mutants
– Permissive temperature
Mutants function normally
– Restrictive temperatures
Mutants function abnormally
Cell fractionation
• Homogenization
• Organelles fractionation by centrifugation