Kontinkangas, L101A
Biochemistry of cellular organelles
Lectures: 1. Membrane channels;
2. Membrane transporters;
3. Soluble lipid/metabolite-transfer proteins;
4. Mitochondria as cellular organelles;
Seminar: Isolation of subcellular organelles;
5. Mitochondrial inheritance;
6. Mitochondria in health and disease;
7. Endoplasmic Reticulum (ER) and lipids;
8. Structure and function of peroxisomes;
Seminar: Mitochondria and other organelles.
Dr. Vasily Antonenkov, Visiting professor
Dept. Biochemistry, Oulu University
Oulu, Finland
Web site:
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Seminar information
• 2 seminars, correspondingly – 2 presentations from each student,
duration of the presentation – 15-20 min;
• The seminars are compulsory!
• Presentations on the seminar: one or two students (not more) for
one report, if two students – each student should present half of the
report;
• Topics of the seminar presentations will be distributed in the break
of the first lecture using lottery system.
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Topics of the first seminar: ‘Isolation of
subcellular organelles’ 26.09.14 (10-12h)
How to isolate:
Focus on:
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• Short description of morphology in vivo
and in vitro and role of the
corresponding organelle in the cell;
• Isolation procedure (in details);
• Characterization of the purity of isolated
organelles;
• Difficulties during isolation;
• What is the reason to isolate organelles
– what we can study using the isolated
fraction and how to do this.
Nuclear (1);
Microsomes (2);
Lysosomes (3);
Golgi complex (4);
Peroxisomes (5);
Plasma membrane (6);
Lipid bodies (7);
Mitochondria (8).
Kontinkangas, L101A.
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Lecture 2: Membrane transporters
Lecture content:
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Examples of membrane channels;
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Aquaporins;
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Flip-flop and transfer of fatty acids;
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Key features of membrane transporters, dependence on energy
consumption;
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How to study membrane transporters;
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Donnan equilibrium;
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Examples of membrane transporters.
KSper’s in sperm membrane
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4 Ksper’s (Potassium sperm
channels) were discovered
using patch clamp in different
parts of spermatozoa. These
channels are unique for
spermatozoa.
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Knock-out of one of these
channels leads to slow
movement of the tail of
spermatozoa that may lead to
male infertility.
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The activity of Ksper’s
channels is changes by pH and
membrane potential that leads
to hyper activated mobility of
spermatozoa.
TRP family of channels
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TRP: transient – most channels are open
not permanently, but at certain time
interval; receptor – contain receptor part in
the molecule, which recognize ligand;
potential – weakly voltage-dependent.
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Mammals contain up to 28 family members
separated into several subfamilies
according to sequence homology.
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Single TRP subunit is made up of 6
transmembrane domains with a pore
constructed by the 5th and 6th segments of
four subunits. Therefore, the hole itself is
between subunits. The channel may be
formed from homo- and heterotetramers.
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The channels are permeable to cations,
including Ca ions.
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Activated by multiple ways including intraand extracellular messages, heat and cold,
chemical compounds, mechanical stimuli,
and osmotic stress.
MRPM8-deficient mice
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Less sensitive to the mild cold:
licking of the paw after
application of acetone less
frequent, do not avoid presence
in the cooled chamber, etc.
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Another channel(s) are
responsible for sensing of the
deep cold.
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How channels sense cold or
heat is not clear – allosteric
transformation of proteins –
similarity to denaturation which
occurs during heating or
cooling.
Aquaporins
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In year 2003 the Nobel prize in chemistry
(not in Physiology as usual for biological
studies) has been awarded for the study of
aquaporins;
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Aquaporins are membrane water channels
controlling the content of water in cells.
Some aquaporins are also permeable for
small sugar-like molecules such as glycerol.
However, they completely impermeable to
charged molecules including protons;
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More than 10 different types of mammalian
aquaporin channels have been identified to
date;
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A single human aquaporin channel facilitates
water transport at a rate of about 3 billion
water molecules per second. Such transport
is bidirectional, in accordance with the
prevailing osmotic gradient.
Aquaporins – mechanism of action
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Aquaporin forms a tetramer in the cellular
membrane, with each monomer acting as a
water channel;
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The narrow shape of the pore allows only
small molecules like water to pass through it;
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Aquaporin molecule consists of 6 alpha
helixes penetrating the membrane plus
several loops. Two of these loops contain
conserved asparagine-proline-alanine (NPA)
motif which helps to form a 3-D hourglass
structure of the channel. In addition,
asparagine is involved in the interaction with
water molecules passing the channel.
Aquaporins – mechanism of action
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Movie – Internet
explorer, favorites,
structure, dinamics and
function of aquaporins:
http://www.ks.uiuc.edu/
research/aquaporins/
The selectivity filter of the
channel is formed by aromatic
amino acids and arginine.
Positively charged arginine acts
to weaken the hydrogen bonds
between water molecules and
also acts to prevent proton
movement across the membrane.
Water molecules move through
the narrow channel by orienting
themselves in the local electrical
field formed by atoms of the
channel wall.
Some lipids are able to form large channels (pores) in
lipid bilayer
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Mitochondrial permeability transition (MPT)
pore that is involved in the apoptosis can be
detected using electrophysiological approach.
Several known proteins are regulators of the
gating of this channel. The gating is also
under control of Ca and some drugs;
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However, the protein nature of the MPT pore
is an enigma. Several candidate proteins
have been rejected after knock-out of them
because this did not lead to abolishing
activity of the MPT channel;
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One idea is that the channel may be not a
protein, but a certain lipid. Ceramide which is
a member of sphingolipid family is able to
form a very large pore in an artificial
membrane. The ceramide channels possess
some characteristics of the MPT pore.
Flip-flop and phospholipid asymmetry in biological
membranes
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Proteins and phospholipids are located in
biological membranes asymmetrically. For
instance, the total amount of phosphocholine
molecules is larger in one leaflet of the
membrane than in the other one;
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Flip-flop is a jump of lipid molecule from one to
another leaflet. It is forbidden for phospholipids
but occurs for medium and long chain free fatty
acids (but not for very long fatty acids);
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Enzymes which catalyze the flip-flop of lipids
are called flippases. Several membrane
transporters may have flippase mechanism of
action (ABC transporters, some ATP-ases,
etc.). Most of them are ATP-dependent
enzymes that ensure transfer of lipids across
the membrane in certain direction, even against
concentration gradient.
How free fatty acids are transported across the
membrane?
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In mammalian organism free fatty acids
(FFA) from blood usually have to overcome
several membrane barriers in the cell to
reach place of their metabolism (i.e.
oxidation in peroxisomes or phospholipid
synthesis in endoplasmic reticulum);
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Most membranes allow rapid flip-flop of
FFA. In addition, some transporters catalyze
this flip-flop. However, the flip-flop in most
cases is a random process, without any
preference to certain direction;
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Acyl-CoA synthases are located only on the
one side of the membrane as peripheral
membrane proteins. They guarantee
unidirectional transfer of fatty acids across
the membrane by coupling them with CoA.
The flip-flop of the resulting product – acylCoA – is forbidden.
Donnan equilibrium
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Donnan effect can explain formation of a
transmembrane pH gradient in the presence of
non-selective channels open to solutes;
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If proteins inside the compartment are charged
mostly negatively, they will attract small
positively charged ions from surrounding
cytoplasm to preserve electroneutrality. This
creates a concentration gradient of small ions
across the membrane;
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pH is the concentration of protons (H+) in a
certain solute which inversely correlates with
the concentration of hydroxyl ions (OH-). These
small ions should follow rules of the Donnan
equilibrium;
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Recently, the pH gradient has been found
across the outer mitochondrial and peroxisomal
membranes. Both these membranes contain
non-selective channels and open to solutes.
Many scientists beleive that if pH
gradient or gradient of any small ion
is registered across the membrane –
this indicates that this membrane is
closed to solutes and should contain
transporters instead of non-selective
channels.
Transporters
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Contrary to channels, transporters
do not form a hole that is open all
the way through the membrane.
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Two possible variants for the action
of transporters: (i) direct movement
of the transporter across the
membrane; (ii) allosteric
transformation leading to binding of
the substrate on one side of the
membrane and releasing of it on the
other side.
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A transporter transfers one or few
solute (lipid) molecules per
conformational cycle, whereas a
single channel opening event may
allow flux of many thousands
molecules.
Movement of transporter across the membrane
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The transmembrane movement was
shown only for some antibiotics
transferring certain ions, e.g.,
valinomycin.
One molecule of valinomycin binds
one ion of dehydrated potassium and
hence shelters it from hydrophobic
environment of the lipid bilayer. The
ring of valinomycin is hydrophobic
outside.
Complex of antibiotic with potassium
is much more soluble in the
membrane lipid bilayer than the ion
itself. This accelerates movement of
the ion across the membrane
according to concentration gradient.
Allosteric transformation of transporters fixed in the
membrane
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Proteins that act as carriers
(transporters) are too large to
move across the membrane,
they are transmembrane
proteins with fixed topology;
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Transporter proteins cycle
between conformations in
which a substrate binding site
faces one side of the
membrane and than the other
side.
How to study activity of transporters?
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Two common experimental systems for
studying the function of transport proteins
are:
(i) liposomes containing purified transport
protein;
(ii) cells transfected with gene coding for a
particular transport protein. The transfected
cells show activation in the transfer of
substrate across the plasma membrane.
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Steps in the liposomal assay:
(i) isolation of transporter;
(ii) formation of proteoliposomes;
(iii) incubation of proteoliposomes with
radioactive substrate;
(iv) separation of proteoliposomes from
unbound substrate molecules;
(v) measurement of radioactivity.
Passive/active transport
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Passive transport takes place only
along the concentration gradient;
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Active transport uses energy to
drive substrate against concentration
gradient;
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Direct active transport: some
transporters bind ATP directly and
use energy of its hydrolysis to drive
transfer of the substrate; these
transporters are all ATP hydrolases.
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Indirect (secondary) active
transport: some transporters use an
energy already stored in the gradient
of a directly pumped ion to drive
transfer of substrate according to
concentration gradient of this directly
pumped ion (co-transporters).
Classes of transporter (carrier) proteins
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Uniport transporters mediate transport
of a single solute (active or passive);
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Symport (co-transport) carriers bind
two dissimilar solutes (A and B) and
transfer them together across a
membrane. Transport of the two solutes
is always coupled. Usually symporters
facilitate indirect active transport;
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Antiport (exchange diffusion) carriers
exchange one solute for another across
a membrane. Most antiporters shows
‘ping-pong’ kinetics: a substrate binds
and is transported, then another
substrate binds and is transported in the
other direction. Only exchange is
catalyzed, not net transport.
Examples of different transporters
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Uniporters: valinomycin; Glut1
transports glucose into most
mammalian cells according to
concentration gradient (passive
transport); ATP-dependent
transporters catalyzing direct
active transport.
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Symporters: glucose-Na symport
in plasma membranes of some
epithelial cells; bacterial lactose
permease catalyzes uptake of the
disaccharide lactose into E.coli
along with proton, the transfer is
driven by proton gradient.
Permease is the old name for
some bacterial transporters.
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Antiporters; adenine nucleotide
translocase (ADP/ATP exchanger)
catalyzes 1:1 exchange of ADP for
ATP across the inner mitochondrial
membrane.
Biomembranes contain different sets of channels and
transporters
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Three types of biological membranes:
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1. Open to all solutes - contain nonselective channels (outer membranes
of gram-negative bacteria and
mitochondria, nuclear membrane).
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2. Open to small solutes only – contain
non-selective channels for small
solutes and specific transporters for
bulky solutes: ATP and some cofactors
(peroxisomal membrane).
a
NADH
Ps
Cyt
ATP
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3. Closed to all solutes – contain
mainly transporters, but also highly
regulated selective channels (inner
membrane of gram-negative bacteria
/E.coli/, plasma membrane, inner
mitochondrial membrane, lysosomes,
endoplasmic reticulum).
Transporter
Uric acid
Glycine
Channel
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Functional cooperation of different transporters
Example: absorption of glucose in small intestine
Glucose is transferred into epithelial cells against concentration gradient by means
of secondary active transport (Glucose-Na sympoter). The driving force is the Na
transmembrane gradient that is created by primary active transport (ATPdependent Na-K antiporter). Glucose leaves epithelial cells along the concentration
gradient by means of passive transport (GLUT2 uniporter).
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Crystal structures of ATP-dependent ion pumps
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The ATP-dependent ion pumps (P-type
ATPases) transfer ions (H, K, Na, Ca)
and some other compounds, including
lipids, against their concentration
gradient using energy of ATP. Some of
them are abundant and important
proteins, like Ca pump in muscles, Na/K
pumps in kidney, etc. Some of these
transporters are involved in maintaining
pH gradients across the membrane.
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The pumps are heteromers of different
subunits.
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Isolation of native proteins from, e.g.,
rabbit or pig; crystallization with different
components (ATP, ions) which gives a
picture of conformational changes of the
proteins.
Na/K-ATPase of plasma membrane – mechanism of action
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The pump, with bound ATP, binds 3 intracellular Na ions;
ATP is hydrolyzed, leading to phosphorylation of the pump and subsequent release
of ADP;
A conformational change in the pump exposes the Na ions to the outside. The
phosphorylated form of the pump has low affinity for Na ions, so they are released;
The same phosphorylated form has high affinity for K ions, so it binds two K ions
that triggers change in conformation and protein dephosphorylation;
The unphosphorylated form of the pump has a higher affinity for Na ions than K
ions, so the two bound K ions are released inside the cell;
New ATP molecule interacts with transporter and process starts again.
The Na/I symporter
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The Na/I symporter mediates active
transport of iodine (I) across plasma
membrane of the thyroid (to form
thyroid hormone) or lactating breast
(to provide iodine with milk).
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It works against concentration
gradient of iodine but along the
gradient of Na.
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It is also active in breast cancer cells
and can be used for treatment with
radioactive iodine like in the case of
thyroid cancer.
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The studies are under way to make
viruses with recombinant symporter
to invade other cancers like
prostate.
ABC transporters
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ATP-binding cassette (ABC)
transporter superfamily. Cassette
means here repeated amino acid
sequence (signature) of the protein.
The transporters can be divided into
two subtypes on the basis of the
direction of the transport reaction.
ABC importers present only in
prokaryotes (~ 80 distinct systems in
E.coli) and require a binding protein.
ABC exporters are found in both,
pro- and eukaryotes. They take their
substrate directly from cytoplasm.
~50 ABC transporters are present in
humans comprising 7 families. The
transporters mainly participate in the
transport of amphipathic lipids
including acyl-CoA’s, cholesterol
and also drugs.
Structure of ABC transporters
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The basic organization of ABC
transporters: two transmembrane
domains (TM) that provide a way for
the cargo and two cytoplasmic
nucleotide-binding domains that bind
and hydrolyze ATP. The membrane
domains may consist of one or two
polypeptide chains.
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TM’s of the ABC exporters always
contain 12 alpha helices (6 in each
subunit). The TM’s of the ABC
importers may contain between 10
and 20 alpha helices (5 to 10 in each
subunit). They mainly consist of two
polypeptide chains but some of them
are formed by only one chain.
ABC transporters – how are they work?
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Binding of ATP produces ‘closing’ of the
gap between nucleotide-binding domains
which triggers flipping of the
transmembrane domains from an inwardfacing to an outward-facing
conformation. ABC importers may now
accept substrates from the opposite
(periplasmic) side of the membrane. ATP
hydrolysis leads to opening of the gap
between nucleotide-binding domains that
allows release of the substrates into
cytoplasm;
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ABC exporters may extrude bound
substrates (lipids or drugs) to the
environment. The mechanism is the
same as for importers, but all steps are
in the opposite direction.
Suggested questions
• Ksper’s channels – what is it?
• TRP family of channels – structure and function;
• What means – structure of water? How this structure is formed?
• Aquaporins – structure, function, and mechanism of action;
• Lipid channels;
• Flip-flop transfer – what is it?
• Function of acyl-CoA synthases;
• Donnan equilibrium – what is it?
• Mechanism of valinomicin action as a transporter;
• How to study activity of transporters?
• Passive and active transport – what is the difference?
• Uniport, symport, and antiport transporters – explain the difference;
• What is the difference in transport mechanisms between outer and inner membranes
of mitochondria or bacteria?
• Cooperation between different transporters in transfer the same metabolite;
• Na/I symporter;
• ATP-dependent transporters – mechanism of action;
• ABC transporters – structure and mechanism of action;