BIO NOTES

Nuclear localization signals sequences direct proteins from cytosol into nucleus. It contains
lysine and arginine (two or one short sequence). Nuclear transport receptors bind to NLS and
help direct the new protein (prospective nuclear protein) to the pore by interacting with the
nuclear pore fibrils. After interacting with the fibril, the structure in the center of the nuclear pore
opens just the right amount to let the protein go in (using GTP hydrolysis). After that the NTR
returns to the cytosol for reuse. Nuclear pores transport proteins in their fully folded shape. Its a
big difference because Proteins unfold during their transportation to other organelles.
Even thought both mitochondria and chloroplast contain their own genome, most of their
proteins are encoded by genes in the nucleus and then they are imported from the cytosol. They
have a signal sequence at the N-terminus that allows them to enter the mitochondria. Those
proteins pass by specialized sites where the two membranes of the organelles are in contact.
Chaperone proteins help pull the proteins across the two membranes and then they help them
fold back once inside the organelles. Signal sequences are cleaved off. Transport to a particular
site ex: the inside or outside membrane require further sorting signal which appear in general
only after the first signal sequence is removed. Membrane phospholipids of mitochondria and
chloroplasts are imported via water soluble lipid carrying proteins (from the ER) that extract
lipids from one membrane to another to ensure characteristic lipid composition of membranes is
retained.
ER serves as an entry point for proteins destined for other organelles (lysosome, endosome,
Golgi apparatus) and also for itself. Once they enter ER from cytosol they continue their journey

inward via transport vesicles from organelle to organelle. Two types of proteins water soluble
proteins which are completely translocated into the ER membrane and released into the lumen
(they are destined for secretion or for the lumen of another organelle) and prospective
transmembrane proteins which are partly translocated in ER membrane and are embedded in it
(they are destined for the ER membrane, the membrane of another organelle or the plasma
membrane). Unlike Mitochondria or chloroplast or peroxisome proteins the proteins that enter
the ER are still being synthesized. Thats why there are ribosomes on the membrane of the ER to
keep synthesizing the molecules. Two types of ribosomes: membrane bound ribosomes in ER for
translocating proteins and free ribosomes that synthesize the rest of the proteins encoded by
nuclear DNA. As an mRNA molecule is translated many ribosomes bind to it forming a
polyribosome. If the mRNA molecule encodes a protein with an ER signal the polyribosome
becomes directed to the ER membrane.

Soluble proteins
The soluble proteins that are surrounded by free ribosomes for translation but they already have a
signal sequence that directs them to the ER need two things to get to the ER: (1) a signal
recognition particle (SRP) and a an SRP receptor. The SRP binds to the signal sequence and
causes the protein translation by the ribosomes to slow down. Once next to the ER, the SRP
binds to the SRP receptor in the ER membrane. The SRP then is released passing the protein to
ribosome to a protein translocation channel then it inserts the polypeptide into the membrane.
The signal sequence which for soluble proteins is almost always at the end terminus functions
also to open the translocation channel.

Transmembrane proteins
Not all proteins that enter the ER are released into the lumen some remain embedded into the ER
membrane. Some parts of the polypeptide must be translocated clear across the lipid bilayer
while others remain fixed in the membrane. For these proteins, the beginning is the same as
soluble proteins with the SRP and SRP receptor. However for these polypeptides there is a
sequence of hydrophobic amino acid called a stop transfer sequence that drifts into the plane of
the lipid bilayer where it forms an alpha helix membrane spanning segment that anchors the
protein in the membrane. N terminal signal sequence is released from channel into the lipid
bilayer and is cleaved off. So at the end we have a trans protein with the N terminus in the
luminal side and the c-terminus on the cytosol. In some proteins, an internal signal sequence is
used not a terminal one and that one never gets cleaved off its called a start transfer sequence
and it give a protein with two parts in the bilayer and a loop in the middle. Look page 511.
Hydrophobic signal sequences work in pairs, an internal start transfer serves to initiate
translocation until a stop transfer sequence is reached. For multipass proteins many start and stop
transfer sequences are added one star translation and the other stops translation and at the end we
will have something looking like a sewing machines work.

Vesicular Transport
ER is only the first destination of certain proteins that are in general going to be
redirected maybe to the Golgi apparatus then to other endomembranes. The proteins are in
general carried into transport vesicles. Two ways: from ER to plasma membrane and from
plasma membrane to lysosomes. Route of communication between the inside and the
surrounding of the cell. That permits cell to undergo chemical modifications such as addition of

carbohydrates (side chains of proteins and lipids) and formation of disulfide bonds that stabilize
protein structures.

Transport vesicles carry proteins between compartments

The route is: biosynthesis of proteins on the ER membrane and their entry in the ER and then it
goes to the Golgi and from there to the cell surface. From the Golgi a side branch leads off
through endosomes to lysosomes look figure page 512. Endocytosis is responsible for ingestion
and degradation of extracellular molecules from plasma membrane through endosome to
lysosome. Each vesicle contains specific proteins and only fuses with specific targets. Ex:
vesicles with proteins that are from the Golgi only fuse with plasma membrane and exclude any
protein that has to stay in Golgi. Each organelle has to keep its own identity.

Coated vesicles:
The coat is there to shape the membrane into a bud and it helps capture molecule for onward
transport. Coated vesicles come from outside and bud into the cytosol or from Golgi going
outside the cell. Ex clathrin coated vesicles bud from Golgi on the outward secretory pathway or
from plasma membrane on the inward endocytic pathway. Clathrin attaches to the bud and
assemble into a basket like network that starts shaping the vesicle. Dynamin (small GTP binding
molecule) forms a ring around the neck of the pit. Together with other proteins it causes the neck
to constrict and it pinches off the vesicle from the membrane.
Look at image in page 514: first we have cargo receptors that bind to specific proteins, and then
we have adaptin which is a coat protein that plays a role in capturing cargo molecules. It also
secures clathrin to the membrane. Cargo molecules possess a specific signal that is recognized by

cargo receptors on the membrane. Since there are two types if clathrin coated vesicles: the one
from the plasma membrane and the one from the Golgi, there are two types of adaptin also: the
one that bind cargo on the plasma membrane and the one that bind cargo molecules from the
Golgi. There are also other coated vesicles called COP coated vesicles which are responsible
from carrying molecules between the ER and the Golgi and other from different parts of the
Golgi. Change in pH causes later the cargo receptors to let go of the molecules. After the budding
each vesicle is transported to destination by motor proteins and they have to fuse to specific
targets. For that reason, there are receptors on the destination membrane that need to recognize
the markers on the surface of the vesicle. There should be recognition and DOCKING of the
receptors. Docking happens when molecules are close together and the proteins at their surfaces
interact. Docking is followed by addition of the new membrane to the old one but it takes more
time because it awaits a signal. DO NOT FORGET disulfide bonds are never in cytosol because
of the reducing environment. PROTEINS IN THE LUMEN OF THE ER NEVER SEE THE
CYTOSOL AGAIN THEY GET CARRIED IN VESICLES.

Secretory pathways
Newly synthesized proteins and lipids, carbohydrates are delivered from the ER via the Golgi to
the cell surface. Proteins as they pass from one membrane to the other are monitored to check
that they folded properly and are assembled with their appropriate partners so that only correct
built proteins are released.
In the ER disulfide bonds are formed and they help stabilize proteins that will undergo pH
changes or degradative enzymes outside the cell. Many proteins that are in the ER get converted
into glycoproteins by covalent attachment of short oligosaccharide side chains (glycosylation). It

is carried out by glycosylating enzymes present in the lumen of the ER. FEW PROTEINS IN
THE CYTOSOL ARE GLYCOSYLATED! The role of oligosaccharides is to protect the protein
against degradation, or hold it in the ER until it is properly folded; they can also guide the
protein to the right organelle by serving as transport signal for packaging the protein into the
appropriate vesicle. Sugars are added by blocks. They are added first to a specialized lipid called
dolichol but then its added at the NH2 end of an asparagine side chain. The addition takes place
in a single enzymatic step catalyzed by a membrane bound enzyme that has its active side
exposed in the luminal side of the ER. The most common type of linkage found on glycoproteins
is the N-linked (oligosaccharide side chains linked to an asparagine NH2).
The proteins that are destined to stay in the ER but escape into the Golgi are brought back thanks
to the ER retention signal a C terminal which is recognized by a membrane. There are chaperon
proteins that are on the ER membrane they check if proteins are folded properly and are with the
good partners. Antibodies that fail to assemble properly are ultimately degraded. In the case of
cystic fibrosis (degeneration of lungs) the plasma membrane transport protein is slightly
misfolded and the ER retains it. The bad thing is that that protein could eventually function
properly as a chloride channel if it got the chance to reach the plasma membrane.

The Golgi apparatus

Its usually located near the nucleus and in animal cell its often close to the centrosome. Its a
collection of flattened membrane enclosed sacs called cisternae. It has a cis face adjacent to the
ER and a Trans face towards the plasma membrane. Proteins travel by budding from one
cisternae and fusing with the other. Proteins move from the cis onward or if the contain protein
that were meant for the ER they move backward. Trans Golgi network sorts proteins that are

destined to the lysosomes and or for the cell surface. Many of the oligosaccharides added
undergo further modifications in the Golgi apparatus. Oligosaccharides are modified as they pass
through the Golgi apparatus. Sugars are removed and added. Depending on the location on the
Golgi enzymes will differ. Enzymes that act early will be in the cis and the one that act late will
be in the trans one.
The constitutive exocytosis pathway consists of buds going from the Golgi directly to the plasma
membrane, it operates continually. It supplies newly made lipids and proteins to the membrane.
Its the pathway for plasma membrane growth, permits the cell to enlarge before dividing.
Constitutive exocytosis doesnt need a signal like the one going to the lysosome. Its a
nonselective pathway (default pathway). Some of the proteins that are secreted stick to the
membrane, other go into the extracellular matrix, some diffuse into the extracellular fluid to
nourish or signal other cells.
Regulated exocytosis pathway operates only for cells specialized for secretion, they produce
large quantities of particular products like mucus, digestive enzymes or hormones which are
stored into secretory vesicles. They bud from the trans golgi network and they accumulate next
to the plasma membrane and wait for the extracellular signal that will stimulate them to fuse with
p membrane. Ex: insulin. Proteins that travel through this pathway have surface properties that
cause them to aggregate under ionic conditions (acidic pH and high ca2+). They are detected by
an unknown mechanism that packages them into secretory vesicles. Constitutive pathway goes
direct doesnt need packaging. The good things about aggregation is that it has the advantage of
making vesicles more concentrated than unaggregat ed protein in the golgi lumen. About 200x
permits more things to be secreted promptly when triggered.

The cytosol is the default location for proteins. Nucleolus is where ribosomal RNA is
transcribed. Proteins can move or rotate but they dont flip flop. Histones are protein beads
around which DNA wraps. Histone + DNA = chromatin. NPC means nuclear pore complexes. A
sphincter (of the nuclear pore) maintains constriction of a passage or orifice and relaxes as
required by normal physiological functioning. Molecules with molecular weight less than 5000
can diffuse through pores. Peroxisomes are organelles that function to rid the cell of toxic
substances. Unlike lysosomes which are formed by secretory pathway, peroxisomes enlarge and
divide. The signal sequences on the proteins going to the nucleus are not cleaved because after
mitosis they will need to reenter the nucleus. Tim and Tom are on the mitochondria: one is the
translocator in the inner membrane and the other is the translocator in the outer membrane. First,
proteins interact with Tom which ushers them to the pore formed by another Tom, then enter the
matrix using the pore complex made of Tim which is in the inner membrane. TOM complex will
include import receptors that initially recognize the signal sequence. Proteins that go to
chloroplast, mitochondria and peroxisome dont go through the ER. Hydrogen that are being
pumped into the matrix cause a potential gradient more negative in the matrix and that favores
proteins being pumped inside the matrix (electrochemical proton gradient). Role of ER is also
detoxification of water insoluble drugs. Proteins are threaded through translocation channels.
What cause the vesicles that accumulate next to the membrane (in regulative exocytotic
pathway) to fuse is a rise in the level of Ca2+. The signal of insulin is glucose. Scramblase is an
enzyme that causes the flip flopping in random order of the phospholipids. Flipase however
selects which one to flip. Flippasesare enzymes located in the membrane responsible for aiding
the movement of phospholipid molecules between the two leaflets that compose a cell's
membrane (transverse diffusion). Phospholipid molecules that are synthesized in the cell are

incorporated into the cytoplasmic face of the membrane, where flippases can transfer them to the
exoplasmic face. Many cells maintain asymmetric distributions of phospholipids between their
cytoplasmic and exoplasmic membrane leaflets.[1] The loss of asymmetry, particularly the
appearance of the anionic phospholipid phosphatidylserine on the exoplasmic face, can serve as
an early indicator of apoptosis. Apoptosis is a programmed cell death that is carried out safely to
dispose of the corpse and fragments. Its different from necrosis which is a programmed cell
death due to an acute cellular injury. Scramblase is responsible for the transportation of
phospholipids across the bilayer. Inactivation of flipase cause permanent activation of scramblase
and that causes cell death. Pinocytosis is cold cellular drinking and phagocytosis is cellular
eating. Phagocytosis involves the ingestion of large particles like cell debris bigger than 250nm.
Some cells cant eat big cells they need to break it down with extracellular enzymes before
taking them up. Phagocytic cells like the white blood cells protect us against invading
microorganisms. Binding of antibody coated bacteria to receptors causes phagocyte cells to
engulf the bacterium by pseudopods. Mycobacterium of tuberculosis inhibits the fusion of the
phagosome with the lysosome so the cell divides with it. Phagocytic cells scavenge injured and
dead cells. Pinocytosis is used by some cells to ingest their own plasma membrane (they remove
their own plasma membrane). Pinocytosis is in general done by clathrin coated pit which later
shed the clathrin and fuse with an endosome. Pinocytosis balances fluid intake and fluid loss.
Receptor mediated endocytosis involves uptake of macromolecules. It provides a selective
concentrating mechanism that increases the internalization of macromolecules 1000 folds
without taking large volumes of extracellular fluid: Example cholesterol uptake. Cholesterol is
transported in the blood under the form of particles called low density lipoproteins LDL that bind
to cell surface. The acidic interior of the endosome cause the LDL to dissociate from the

receptor. Then receptors are returned in transport vesicles for reuse and the LDL is delivered to
the lysosome. LDL is then broken down by hydrolytic enzymes and the cholesterol escapes then
in the cytosol foruse. R mediated pinocytosis causes uptake of vitamins and irons. Endosomal
compartment is made of a network of interconnected tubes and vesicles. The early endosome is
near the plasma membrane and the late endosome is near the nucleus. Interior of endosome kept
acidic by ATP driven pump (proton). Endosome plyas a role of sorting in the endocytic pathway
but the Trans golgi does it for the exocytotic one. There are transport proteins in the lysosome
that transport digested macromolecules to places where they can be reused. The enzymes and
membrane proteins of the lysosome are made in the ER and during their transport from ER to the
golgi they are tagged with a phosphorilated sugar that causes them to go to the lysosome via late
endosome. Phagosomes fuse directly with the lysosome but pynocytotic products fuse with
lysosome via endosome. V snares and t snares are proteins on the vesicle or target membrane that
mediate docking and fusion. Types of enzyme are: proteases from polypeptides to amino acids,
nucleases for DNA to nucleotides and glycosidases from oligosaccharides to saccharides.
Autophagy helps prevent cancer, neurodegenerations and increases longevity. Its more common
in newborns. RAN GTP produces energy for nuclear exports. RAN is necessary. In some cases
adaptin only recognizes receptors once the cargo is attached (configurational changes)
ACTIN FILAMENTS
Found in all eukaryotic cells and permit phagocytosis, crawling, division, and movement
involving especially the cell surface. They can form stiff and relatively structures like microvilli
or contractile bundles that can contract and act as the muscles of the cell. Actin filaments are
associated to many actin binding proteins that permit them to serve a variety of functions. Actins
form the ring during the last stage of cell division (it forms a ring) and it also forms the leading

edge of a crawling fibroblast. Actin filaments are very thin. They are made of a twisted chain of
globular actin molecules; it has a polarity like the microtubules. They are more numerous,
shorter and more flexible than the microtubules. The total length of actin in the cell is more than
the total length of microtubules in the cell. Actin filaments are rarely isolated most of the time
they are in bundles or networks cross linked. Unlike tubulin, actin monomers are bound to ATP
which is hydrolysed to ADP as soon as the monomer is added. Just like tubulins, ADP decreases
stability and promotes depolymerization. Just like tubulins, actin filaments need to be able to
polymerize and depolymerize fast. So adding toxins produced by marine sponges and fungi can
perturb actin filaments. Fo example cytochalasins prevent actins polymerization and
jasplakinolides stabilize it against depolymerization. Those toxins will stop cell movement
instantly. So dynamic equilibrium between the filaments and the pool of actin monomer is
needed for the cell to move. Unlike tubulin, the concentration of actin monomers in the cell is
much higher than what is required for them to polymerize in vitro. That is because there are
small proteins like thymosin and profiling that bind to actin monomers and prevent them from
polymerizing. By keeping monomers in reserve thymosin plays a crucial role in regulating actin
polymerization. There are also actin binding proteins that bind to actin when its assembled. For
example actin building proteins hold bundles parallel in microvilli , cross linking proteins keep
actin in a gel like meshwork in the cell cortex. Some actin severing proteins like gelsolin
fragment actin into smaller filaments so that the gel like substance will become more fluid.
Actin filaments can also be associated with motor proteins and form tracks along which they can
travel (especially in plant cells). Cell cortex is the layer right underneath the plasma membrane.
Actin filaments govern the shape and mechanical properties of the plasma membrane and the cell
surface. The cell has three steps when its moving: first it pushes a protrusion at its front, then the

protrusion adheres to the surface over which the cell is growing, then the rest of the cell drags
itself towards the front. There are lamelipodia which are sheet like protrusions due to actin
polymerization and then there are filipodia both are exploratory. Axons use their filipodia to
probe the environment to find their path. Nucleation is the formation of the actin filaments.
Some accessory proteins help nucleate actin at the plasma membrane: for example Actin related
proteins ARPs promote formation of branched actin filaments. They add on one side of the actin
and cause it to branch like a tree. That network undergoes assembly in the front and disassembly
in the back. When the network finds a good surface, they bind to a transmembrane protein
integrins which adhere to molecules on the extracellular surface or on the surface of other cells
on which the cell is moving. Meanwhile in the inside of the cell integrins capture actin filaments
creating a good anchorage. To drag the rest of the cell forward it uses internal contractions. All
actin motor proteins belong to the family of myosins. They bind to ATP which gives them energy
for their movement. There are Myosin 1 and myosin 2. Myosin 1 has one head and one tail. The
head interacts with the actin filament and it has an ATP hydrolyzing motor activity that permits it
to move along the filament by binding, detaching and rebinding. The tail can be carrying vesicles
or plasma membrane and move it relative to the cortex to change its shape. Activity of the
accessory proteins can be regulated by external signals so that cell can change shape depending
on its environment. For example receptor proteins embedded in the plasma membrane can be
activated causing all the signals to converge inside the cell on a group of closely related
monomeric GTP binding proteins called Rho protein family. They can cycle between active GTP
bound state or inactive GDP bound state. Acitvation of GTP binding protein CDc42 tirggers actin
polymerization to form filipodia whereas activation of Rac GTP binding protein promotes the
formation of lamelipodia. Those molecular switches (Rho) interact with protein kinases and

accessory proteins that control actin organization. For example Cdc42 enhance actin nucleating
activity by ARP same for Rac but with Rac it also enhances uncapping at the plus end of actin
filaments. Uncapping provides additional sites for actin assembly at plasma membrane causing
formation of large lamelipodia.
Contraction most of the time depend on the interaction between actin and myosin. Myosin two
has two ATPase heads and a long rod like tail. They are the one present in muscles. Myosins two
are dimmers that are made of two identical myosin molecules held together by the tail. Cluster of
myosins bind together by their coiled coil tail and they look like two headed arrow with the
globular heads sticking out. Part of the globular heads point one direction to move the actin that
direction and the other part points towards another direction to move the actin towards another
directions. The effect is to slide sets of oppositely directed actin filaments past one another. That
is seen in contractile bundles and muscle contraction and contractile ring that pinches a dividing
cell in two. Free diffusion is slower than transport along microtubules. The skeletal muscle cells
are a large cell made of the fusion of many small cells. Their nuclei are retained right beneath the
plasma membrane. Their cytoplasm is made of myofibrils which are contractile elements of the
muscle. A myofibril is made up of tiny contractile units called sarcomeres. Sarcomeres cause the
muscle to seem striated. They are organized and made up of actin filaments and filaments of
muscle specific myosin II (thick filaments). The actin filaments extend until the end of the
muscle and they are anchored by their plus end to the Z disc and overlap with myosin filaments.
Contraction is caused by sliding of actin and myosin filaments past each other. That in turn is due
to the interaction between the myosin head (when stimulated) and the actin filaments. The
Myosin head binds to the actin and the ATP is hydrolysed causing conformational change in the
myosin that causes the head to move around 5 nm for each cycle. Each filament is made up of

more than 300 myosin heads and they attach and detach more than five times every second. The
myosins move towards the plus end of the actin filaments.

Muscle contractions are triggered by rise in Ca2+. First signal is received from a nerve terminal.
The excitation spreads in milliseconds into a series of membranous tubes called transverse
tubules that extend inward from the plasma membrane around each myofibril. Then the signal is
relayed to the sarcoplasmic reticulum which is an adjacent sheat of interconnected flattened
vesicles that surround each myofibril like a net. The sarcoplasmic reticulum is a region of
endoplasmic reticulum in muscle cell that contains high concentration of calcium and in response
to the excitement (electrical change in voltage) much of the calcium is released through ion
channels. Ca is a lot used as a signal to relay a message from the exterior to the internal
machinery of the cell. Ca will interact with molecular switches made of specialized accessory
proteins closely associated with the actin filaments. Like tropomyosin which is a rod shaped
molecule that binds to the helix of actin and overlaps 7 actin monomers and prevents the myosin
from associating with the filaments. The other protein is troponin which has a ca sensitive
protein (troponin-C). Troponin is associated with the end of a tropomyosin molecule. When level
of Ca rises, ca binds to troponin and induces a change in shape of troponin and that in turn causes
tropomyosinmolecules to shift their position allowing myosin heads to bind to the actin filament
and cause contraction. The increase in ca stops as soon as the nerve signal stops and ca is rapidly
pumped back into the sarcoplasmic reticulum and troponin and tropomyosin return back to their
original position. Look at page 604. Myosin II in non muscle cells is also activated by rise in ca
but for that type, rise in ca will cause myosin to be phosphorilated and that alters the myosin
conformation and unable it to interact with actin. The same activation happens with smooth

muscles in uterus, arteries etc This kind of activation causes slower and more sustained
contractions. The reason for that is that enzymes are needed to diffuse to myosin heads and to
carry out phosphorilation and dephosphorilation. However this mechanism is less specialized and
can be triggered by variety of signals like adrenaline, prostaglandine etc