TRANSPORT OF SOLUTES

COMPARTMENTALIZATION
IN CELLS

Figure 12-1 The major intracellular compartments of an animal cell

The cytosol (gray), endoplasmic reticulum, Golgi apparatus, nucleus, mitochondrion, endosome, lysosome,
and peroxisome are distinct compartments isolated from the rest of the cell by at least one selectively
permeable membrane.
 Cell Membrane

A431 cell line stained for plasma RT4 cell line stained for plasma U251 MG cell line stained for
membrane (green) membrane (green) plasma membrane (green)

Human Atlas antibodies inc

RELATIVE MEMBRANE TYPE COMPARISON IN TWO DIFF CELLS
Solute transport across membranes

Watson, Essays in Biochemistry, 2015

DIFFUSION THROUGH
LIPID LAYER

DIFFUSION THROUGH
CHANNELS

FACILITATED
TRANSPORT
Direct Active Transport
1. The Na+/K+ ATPase
The cytosol of animal cells contains a concentration of potassium ions (K+) as much as 20 times higher than that in the
extracellular fluid. Conversely, the extracellular fluid contains a concentration of sodium ions (Na+) as much as 10 times
greater than that within the cell.
These concentration gradients are established by the active transport of both ions. And, in fact, the same transporter, called
the Na+/K+ ATPase, does both jobs. It uses the energy from the hydrolysis of ATP to actively transport 3 Na+ ions out of the
cell for each 2 K+ ions pumped into the cell.
•This accomplishes several vital functions: It helps establish a net charge across the plasma membrane with the interior of
the cell being negatively charged with respect to the exterior. This resting potential prepares nerve and muscle cells for the
propagation of action potentials leading to nerve impulses and muscle contraction.
•The accumulation of sodium ions outside of the cell draws water out of the cell and thus enables it to maintain osmotic
balance (otherwise it would swell and burst from the inward diffusion of water).
•The gradient of sodium ions is harnessed to provide the energy to run several types of indirect pumps.
The crucial roles of the Na+/K+ ATPase are reflected in the fact that almost one-third of all the energy generated by the
mitochondria in animal cells is used just to run this pump.
2. The H+/K+ ATPase
The parietal cells of your stomach
use this pump to secrete gastric
juice.
These cells transport protons (H+)
from a concentration of about 4 x
10-8 M within the cell to a
concentration of about 0.15 M in
the gastric juice (giving it
a pH close to 1).
3. The Ca2+ ATPases
A Ca2+ ATPase is located in the plasma membrane of all eukaryotic cells. It uses the energy provided by one molecule of
ATP to pump one Ca2+ ion out of the cell. The activity of these pumps helps to maintain the ~20,000-fold concentration
gradient of Ca2+ between the cytosol (~ 100 nM) and the ECF (~ 20 mM).
In resting skeletal muscle, there is a much higher concentration of calcium ions (Ca2+) in the sarcoplasmic reticulum than
in the cytosol. Activation of the muscle fiber allows some of this Ca2+ to pass by facilitated diffusion into the cytosol where
it triggers contraction.
After contraction, this Ca2+ is pumped back into the sarcoplasmic reticulum. This is done by another Ca2+ ATPase that uses
the energy from each molecule of ATP to pump 2 Ca2+ ions.
• 4. ABC Transporters
•ABC ("ATP-Binding Cassette") transporters are transmembrane proteins that expose a ligand-binding domain at one
surface and a ATP-binding domain at the other surface.
• The ligand-binding domain is usually restricted to a single type of molecule.
• The ATP bound to its domain provides the energy to pump the ligand across the membrane.
• The human genome contains 48 genes for ABC transporters.

• CFTR — the cystic fibrosis transmembrane conductance regulator

•TAP, the transporter associated with antigen processing,
• ABC transporters that pump chemotherapeutic drugs out of cancer cells thus reducing their effectiveness.
• ABC transporters must have evolved early in the history of life. The ATP-binding domains in archaea, eubacteria,
and eukaryotes all share a homologous structure, the ATP-binding "cassette".
Indirect Active Transport
Indirect active transport uses the downhill flow of an ion to pump some other molecule or ion against its gradient.

The driving ion is usually sodium (Na+) with its gradient established by the Na+/K+ ATPase.
Symport Pumps
In this type of indirect active transport, the driving ion (Na+) and the pumped molecule pass through the membrane pump in
the same direction.

•The Na+/glucose transporter. This transmembrane protein allows sodium ions and glucose to enter the cell together.
•The sodium ions flow down their concentration gradient while the glucose molecules are increased.
•Later the sodium is pumped back out of the cell by the Na+/K+ ATPase.
•The Na+/glucose transporter is used to actively transport glucose out of the intestine and also out of the kidney tubules and
back into the blood.
•All the amino acids can be actively transported, for example out of the kidney tubules and into the blood, by sodium-driven
symport pumps.
•Sodium-driven symport pumps also return neurotransmitters to the presynaptic neuron.
•The Na+/iodide transporter. This symporter pumps iodide ions into the cells of the thyroid gland (for the manufacture of
thyroxine) and also into the cells of the mammary gland
•The permease encoded by the lac operon of E. coli that transports lactose into the cell.
Antiport Pumps
• In antiport pumps, the driving ion (again, usually sodium) diffuses through the pump in one direction
providing the energy for the active transport of some other molecule or ion in the opposite direction.
• Example: Ca2+ ions are pumped out of cells by sodium-driven antiport pumps
• Antiport pumps in the vacuole of some plants harness the outward facilitated diffusion of protons
(themselves pumped into the vacuole by a H+ ATPase) to the active inward transport of sodium ions.

• This sodium/proton antiport pump enables the plant to sequester sodium ions in its vacuole.
•Transgenic tomato plants that overexpress this sodium/proton antiport pump are able to thrive in saline
soils too salty for conventional tomatoes.
•to the active inward transport of nitrate ions (NO3−).
A growing number of human diseases have been discovered to be caused by inherited mutations in genes
encoding channels.
•Chloride-channel diseases
• cystic fibrosis;
• inherited tendency to kidney stones (caused by a different kind of chloride channel than the one
involved in cystic fibrosis).
•Potassium-channel diseases
• the majority of cases of long QT syndrome, an inherited disorder of the heartbeat;
• a rare, inherited tendency to epileptic seizures in the newborn;
• several types of inherited deafness.
•Sodium-channel diseases
• inherited tendency to certain types of muscle spasms;
• Liddle's syndrome. Inadequate sodium transport out of the kidneys, because of a mutant sodium
channel, leads to elevated osmotic pressure of the blood and resulting hypertension (high blood
pressure).
OMIM
Endocytosis and Exocytosis
• Endocytosis and exocytosis are the processes
by which cells move materials into or out of the
cell that are too large to directly pass through the
lipid bilayer of the cell membrane.
• Large molecules, microorganisms and waste
products are some of the substances moved
through the cell membrane via exocytosis and
endocytosis.
• Vesicle exocytosis is a fundamental cellular
process that regulates many biological events,
such as the release of neurotransmitters,
hormones, and cytokines and delivery of proteins
and lipids to the plasma membrane for cell
repair, growth, migration, and regulation of cell
signaling
Endocytosis serves many purposes, including:
•Taking in nutrients for cellular growth, function and repair: Cells need materials like proteins and lipids to
function.
•Capturing pathogens or other unknown substances that may endanger the organism: When pathogens like
bacteria are identified by the immune system, they are engulfed by immune cells to be destroyed.
•Disposing of old or damaged cells: Cells must be safely disposed of when they stop functioning properly to
prevent damage to other cells. These cells are eliminated through endocytosis.

Types of endocytosis
There are two types of endocytosis: phagocytosis and pinocytosis.
Phagocytosis
• Phagocytosis (the condition of “cell eating”) is the process by which large
particles, such as cells or relatively large particles, are taken in by a cell.
• In preparation for phagocytosis, a portion of the inward-facing surface of the
plasma membrane becomes coated with a protein called clathrin, which
stabilizes this section of the membrane.
• The coated portion of the membrane then extends from the body of the cell and
surrounds the particle, eventually enclosing it.
• Once the vesicle containing the particle is enclosed within the cell, the clathrin
disengages from the membrane and the vesicle merges with a lysosome for
the breakdown of the material in the newly formed compartment (endosome).
• When accessible nutrients from the degradation of the vesicular contents have
been extracted, the newly formed endosome merges with the plasma
membrane and releases its contents into the extracellular fluid.
• The endosomal membrane again becomes part of the plasma membrane.
Pinocytosis
• A variation of endocytosis is called pinocytosis. This literally means “cell drinking” and
was named at a time when the assumption was that the cell was purposefully taking in
extracellular fluid.
• In reality, this is a process that takes in molecules, including water, which the cell needs
from the extracellular fluid.
• Pinocytosis results in a much smaller vesicle than does phagocytosis, and the vesicle
does not need to merge with a lysosome.
• A variation of pinocytosis is called potocytosis.
This process uses a coating protein, called
caveolin, on the cytoplasmic side of the plasma
membrane, which performs a similar function to
clathrin. The cavities in the plasma membrane that
form the vacuoles have membrane receptors and
lipid rafts in addition to caveolin.

• The vacuoles or vesicles formed in caveolae

(singular caveola) are smaller than those in
pinocytosis. Potocytosis is used to bring small
molecules into the cell and to transport these
molecules through the cell for their release on the
other side of the cell, a process called transcytosis.
Receptor-Mediated Endocytosis
• A targeted variation of endocytosis employs receptor proteins in the plasma membrane that
have a specific binding affinity for certain substances.

• In receptor-mediated endocytosis, as in phagocytosis, clathrin is attached to the cytoplasmic

side of the plasma membrane. If uptake of a compound is dependent on receptor-mediated
endocytosis and the process is ineffective, the material will not be removed from the tissue fluids
or blood. Instead, it will stay in those fluids and increase in concentration.
• Some human diseases are caused by the failure of receptor-mediated endocytosis. For
example, the form of cholesterol termed low-density lipoprotein or LDL (also referred to as
“bad” cholesterol) is removed from the blood by receptor-mediated endocytosis.
• In the human genetic disease familial hypercholesterolemia, the LDL receptors are defective
or missing entirely.
• People with this condition have life-threatening levels of cholesterol in their blood, because
their cells cannot clear LDL particles from their blood.
• Although receptor-mediated endocytosis is designed to bring specific substances that are
normally found in the extracellular fluid into the cell, other substances may gain entry into
the cell at the same site.
• Flu viruses, diphtheria, and cholera toxin all have sites that cross-react with normal
receptor-binding sites and gain entry into cells.
Exocytosis

The steps of exocytosis

1.A vesicle is formed, typically within the endoplasmic reticulum and the
Golgi apparatus or early endosomes.
2.The vesicle travels to the cell membrane.
3.The vesicle fuses to the plasma membrane, during which the two
bilayers merge.
4.The vesicle’s contents are released into the extracellular space.
5.The vesicle either fuses with or separates from the cell membrane.
Exocytosis
Exocytosis serves the following purposes:
•Removing toxins or waste products from the cell’s interior: Cells create
waste or toxins that must be removed from the cell to maintain homeostasis. For
instance, in aerobic respiration, cells produce the waste products carbon dioxide
and water during ATP formation. Carbon dioxide and water are removed from
these cells via exocytosis.
•Facilitating cellular communication: Cells create signaling molecules like
hormones and neurotransmitters. They are delivered to other cells following their
release from the cell through exocytosis.
•Facilitating cellular membrane growth, repair, signaling and
migration: When cells absorb materials from outside the cell during endocytosis,
they use lipids and proteins from the plasma membrane to create vesicles. When
certain exocytotic vesicles fuse with the cellular membrane, they replenish the cell
membrane with these materials.
Types of exocytosis
• Regulated Exocytosis

• Most exocytotic vesicles contain substances created within the endoplasmic reticulum for use
elsewhere in the body, such as neurotransmitters or hormones. These molecules are then
packaged within a layer of membrane called a vesicle.

Once excreted from the endoplasmic reticulum, these vesicles are transported to the Golgi
apparatus (also known as the Golgi complex) for further modification. The molecules are then
packaged once again in a vesicle that makes its way to the plasma membrane.

The release of these molecules from the cell is termed regulated exocytosis because the
expulsion of the materials is controlled, or regulated, by extracellular signals that cause
membrane depolarization.
• Constitutive Exocytosis
• Constitutive exocytosis, in contrast, doesn’t require any extracellular signals. The majority
of molecules traveling to the plasma membrane do so using this pathway.

• After exocytosis, some exocytotic vesicles are incorporated into the plasma membrane
(full vesicle fusion), while others return to the interior of the cell after their contents have
been released (this is termed the “kiss-and-run” pathway). Others remain docked to the
membrane, where they can be used multiple times (the “kiss-and-stay” pathway).
•
Exocytosis and Endocytosis

Liang et al., Front. Mol. Neurosci., 2015

 Methods of Transport, Energy Requirements, and Types of Material Transported
Transport Method Active/Passive Material Transported
Small-molecular weight
Diffusion Passive
material
Osmosis Passive Water
Sodium, potassium, calcium,
Facilitated transport/diffusion Passive
glucose
Primary active transport Active Sodium, potassium, calcium
Secondary active transport Active Amino acids, lactose
Large macromolecules, whole
Phagocytosis Active
cells, or cellular structures
Small molecules
Pinocytosis and potocytosis Active
(liquids/water)
Receptor-mediated Large quantities of
Active
endocytosis macromolecules
Waste materials, proteins for
Exocytosis Active the extracellular matrix,
neurotransmitters
• Figure 12-6A simplified “roadmap” of protein traffic
• Proteins can move from one compartment to another by gated
transport (red), transmembrane transport (blue), or vesicular
transport (green).
• The signals that direct a given protein's movement through the
system, and thereby determine its eventual location in the cell,
are contained in each protein's amino acid sequence.
• The journey begins with the synthesis of a protein on
a ribosome in the cytosol and terminates when the final
destination is reached. At each intermediate station (boxes), a
decision is made as to whether the protein is to be retained in
that compartment or transported further.
• In principle, a signal could be required for either retention in or
exit from a compartment.
Figure 12-8
Two ways in which a sorting signal can be built into a protein
(A) The signal resides in a single discrete stretch of amino acid sequence, called a signal sequence, that is
exposed in the folded protein. Signal sequences often occur at the end of the polypeptide chain (as shown),
but they can also be located internally.
(B) A signal patch can be formed by the juxtaposition of amino acids from regions that are physically separated
before the protein folds (as shown). Alternatively, separate patches on the surface of the folded protein that are
spaced a fixed distance apart can form the signal.
 GFP tagged protein, protein has to be localized in nucleus

Cloning of gene along with sequence for NLS Cloning of gene with NLS
sequence with GFP

Vector backbone with GFP

Transfection: Genetic component in a vector, into mammalian cells

Lentiviral/Retroviral transduction

Liposomes, lipid based transfection, introduce into cells: empty vector and cloned construct

Electroporation
Figure 12-4
Hypothetical schemes for the evolutionary origins of some membrane-enclosed
organelles

The origins of mitochondria, chloroplasts, ER, and the cell nucleus can explain the
topological relationships of these intra-cellular compartments in euKaryotic cells.

(A) A possible pathway for the evolution of the cell nucleus and the ER. In some bacteria
the single DNA molecule is attached to an invagination of the plasma membrane. Such
an invagination in a very ancient proKaryotic cell could have rearranged to form an
envelope around the DNA, while still allowing the DNA access to the cell cytosol (as is
required for DNA to direct protein synthesis). This envelope is presumed to have
eventually pinched off completely from the plasma membrane, producing a nuclear
compartment surrounded by a double membrane.

As illustrated, the nuclear envelope is penetrated by communicating channels called

nuclear pore complexes. Because it is surrounded by two membranes that are in
continuity where they are penetrated by these pores, the nuclear compartment is
topologically equivalent to the cytosol; in fact, during mitosis the nuclear contents mix
with the cytosol. The lumen of the ER is continuous with the space between the inner
and outer nuclear membranes and topologically equivalent to the extracellular space.

(B) Mitochondria (and plastids) are thought to have originated when a bacterium was
engulfed by a larger pre-euKaryotic cell. They retain their autonomy. This may explain
why the lumens of these organelles remain isolated from the membrane traffic that
interconnects the lumens of many other intracellular compartments.