Made by: Dalia Aljayar

Edited by: Basel jobair

Ribosomes synthesize proteins

ONLY 1 kind of ribosome with a large and a small subunit, how does the protein know where it
should end up? In the nucleus or mitochondria etc.

How are the proteins sorted?

Endomembrane system
Endomembrane system: consists of 1- ER, 2- the Golgi apparatus, 3- Lysosomes, 4- Endosomes
and 5- Secretory Vesicles.
They are involved in the processing of proteins for export from the cell, proteins destined for
lysosomes, and proteins entering the cell.
All Proteins are synthesized in the cytosol by ribosomes, if they’re destined to lysosome or
secretory then they’ll enter the ER.
Once proteins enter the ER they never return to the cytosol. they are carried via vesicle
transport to the other compartments of the system.
Made by: Dalia Aljayar
Edited by: Basel jobair

protein sorting AKA protein targeting: the process of directing proteins to organelles such as
the nucleus, mitochondria, and ER.

I. THE SECROTORY, LYSOSOMAL, AND THE MEMBRANE PATHWAY

- MRNA exists a nucleus

- a ribosome attaches to it

- the first protein has a portion known as a signal peptide AKA signal sequence, once it emerges
from the ribosome then translation (protein synthesis) stops

• The signal-recognition particle (SRP), a ribonucleoprotein with GTPase activity, binds the
signal sequence
• binding to SRP stops translation and directs the ribosome protein complex to the ER
• Why does it stop? Waiting to drag the ribosome-protein complex to the ER

SRP drags ribosome protein complex to ER

on the ER there is SRP receptor, the SRP attaches to the SRP-receptor which causes hydrolysis
of GTP because SRP also has GTPase activity
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Edited by: Basel jobair

The energy from the Hydrolysis of GTP is used to move the ribosome with the signal sequence
onto a channel sitting in ER called translocon the hydrolysis of GTP allows SRP to let go of the
signal sequence

• When ribosome attaches to the translocon there is hydrolysis of TP

protein synthesis takes place again

the protein enters via the translocon, the signal peptidase enzyme inside the ER sees the signal
sequence coming into ER and it chops the signal off

the growing protein now has entered the ER.

:

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Once you enter ER you can only leave via vesicle
proteins entering the secretory or lysosomal pathway are first synthesized on free ribosomes in
the cytosol. These proteins are then transferred to the ER to continue synthesis.

The signal sequence (for the ER) is a series of 9 to 12 hydrophobic amino acids, at the N-
terminal end of the primary protein structure, that identifies the nascent (first part ) as one that
must cross the ER membrane.
Signal sequences mark proteins for translocation (the movement of something from one place
to another) across the ER membrane into the cisterna of Golgi.
Signal sequences table “Dr. rasool said we must know them”

II. NUCLEUS PATHWAY

have a signal sequence BUT it makes full protei n in cytoplasm (no stopping)
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Edited by: Basel jobair

Protein has signal sequence NLS (nuclear localization signal) which attaches to the nuclear
importin receptor (NIR) this brings in the protein inside the nucleus through the gated nuclear
pore
Ran-GTP binds to importin - releasing protein to nucleoplasm (cytoplasm of nucleus)
Ran-GTP bound to the NIR leaves the nucleus.

Hydrolysis of Ran-GTP to Ran-GDP releases NIR.

https://www.youtube.com/watch?v=ZGPpKk-6-K0
ALL proteins are first made on ribosomes that are FREE in the CYTOPLASM aka cytosol (not
attached to ER)
Proteins that go to ER to continue synthesis: are secretory, lysosome or plasma membrane
proteins
proteins used inside the cell, in the cytoplasm or organelles in the cell like peroxisome,
mitochondria, and nucleus are made fully in cytoplasm
NOTE: every organelle has a different signal sequence. Signal sequence determines whether it’ll
be co or post translational and where it is going to end up.

III. MITOCHONDRIAL PATHWAY

proteins are synthesized in the cytosol and recognized by receptors on the surface of
mitochondria.
The whole protein can’t be bought in, the channel isn’t big enough so can’t bring it in as a
tertiary structure

How do we bring it in? HSP 70, a chaperone, unfolds a protein into a straight chain to help bring
it in.
1. the protein is bound to hsp70. The protein’s mitochondrial signal sequence inserts into
an import receptor of the TOM complex in the outer membrane (translocase of outer
membrane, a channel) (TOM20).

Transfer from receptor into tom channel needs ATP

2. Hsp70 dissociates due to ATP hydrolysis. (hydrolysis also helps pull protein in)
3. Protein now goes through tom.
4. the Intermembrane space (between the 2 membranes of the mitochondria) has tons of
h ions, highly charged (all the hydrophobics want to run away from h) so they go
through Tim (translocase inner membrane, a channel)
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Edited by: Basel jobair

Insertion of the protein into the outer membrane (via TOM22, an outer membrane channel)
puts signal sequence in position to interact with TIM complex (TIM23, an inner membrane
channel)
The hydrolysis of ATP and the intermembrane’s charged environment drives proteins to go into
Tim

Translocases in the outer (TOM) and inner (TIM) membrane of mitochondria facilitate the
import. Basically, the TOM and TIM channels allow the protein to go through the outer and
inner membrane.
Where do they get the energy to do so? Hydrolysis of ATPs and the membrane potential
Potential across the inner membrane drives protein into inter-membrane space.

5. Mitochondrial Hsp70 binds to protein in the matrix and uses the energy of ATP
hydrolysis to pull the rest of the protein through.
6. Signal sequence is cleaved off by MPP (mitochondrial processing peptidase).
7. Hsp70 in matrix helps protein to fold back to its tertiary structure

Chaperones (hsp70) now assist in the folding and assembly of mitochondrial proteins back into
their native, three-dimensional structures. Protein folds into correct conformation in
mitochondrial matrix.
NOTE: Chaperone Hsp70 helps at different stages in different ways don’t get confused. initially
it unfolded the protein to help it go through, since the protein is too big, then Hsp70 was
removed. It was used again, now from the inside of the mitochondria, to help pull the rest of
the protein in. there, it was also used to help fold the protein back to its tertiary structure.
: https://www.youtube.com/watch?v=J9EWTI5m2mM

Two pathways are used to sort proteins:

Made by: Dalia Aljayar
Edited by: Basel jobair

1. secretory, membrane, and lysosomal pathway: proteins are inserted into

the ER membrane co-translationally.

The picture shows you that the protein (the blue bit)
still attached to its ribosome (green) and not fully
synthesized attaches to ER (grey) via the signal
sequence (red) to continue synthesis

2. mitochondrial, nucleus, peroxisome pathway: completed proteins are

delivered to the target organelle after translation - post-translational

The protein (blue) is fully synthesized and released

from ribosome (light green) into cytoplasm (it
became dark green) then the fully synthesized
protein attaches to its target organelle (grey) via
signal sequence (red)
Made by: Dalia Aljayar
Edited by: Basel jobair

Glycoproteins
Glycoproteins: large protein molecules with carbs attached to it or oligosaccharides anything
between 3 to 11 carb units
Glycosylation: the process of putting carbs on proteins

Why glycosylation?
Helps in proper protein folding.
Provides protection against proteases (e.g. lysosomal membrane proteins)
for signaling.

Most soluble and membrane-bound proteins made in the ER are glycoproteins, in contrast to
cytosolic proteins.
Glycoprotein synthesis is a 3-part process:
Assembly of the precursor oligosaccharide (the 3-22 carb units)
En-bloc transfer of oligosaccharide to the protein

Modification of the oligosaccharide by removal/addition of sugars

can attach the carb to protein in 2 ways:
The addition of carbohydrates (glycosylation) is the main modification to proteins.
Some glycosylation occurs in the lumen of the ER; others, in the lumen of the cis-,medial-, or
trans-Golgi cisternae
O-linked oligosaccharides: are linked to the hydroxyl group of serine or threonine via N-
acetylgalactosamine.
O-linked oligosaccharides are generally short, often containing only one to four sugar
residues.
In all N-linked oligosaccharides: N-acetylglucosamine (GlcNAc) is linked to the amide nitrogen
of asparagine.
N linked are generally bigger than o linked

N-linked oligosaccharides always contain mannose as well as N-acetylglucosamine and

terminate with a negatively charged sialic acid (NANA N-acetylneuraminic acid) residue.
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How does the attachment take place for n linked?

The core oligosaccharide is built up by the addition of monosaccharide units.
1,2. The first steps occur on the cytosolic face of the ER.

Dolichol: is a fatty acid chain sitting across the membrane of ER contains phosphate and is
facing the cytoplasm
attach to the phosphate of dolichol, two n acetyl glucosamine
mannose, 5 of them, attach to dolichol

Translocation moves the incomplete oligosaccharide across the membrane.

Dolichol instead of facing cytoplasm flips over and faces the cisternal side of ER, the intra side
4. Completion of the core oligosaccharide occurs within the lumen of the ER. The precursors
that contribute additional mannose and glucose residues to the growing oligosaccharide in the
lumen are dolichol phosphate derivatives.

5,6. The core oligosaccharide is transferred from dolichol phosphate to an Asn (asparagine)
residue of the protein within the ER lumen.
oligoside transferase cuts the dolichol and its phosphate. The oligosaccharide is transferred to
asparagine, the asparagine becomes part of protein

The core oligosaccharide is then further modified in the ER and the Golgi complex in pathways
that differ for different proteins.
7. The five sugar residues shown surrounded by a beige screen (after step 7) are retained in the
final structure of all N-linked oligosaccharides. Never affected, always be there

8. The released dolichol pyrophosphate is again translocated so that the pyrophosphate is on

the cytosolic face of the ER.
9. Then a phosphate is hydrolytically removed to regenerate dolichol phosphate.

Blood groups
The human A, B, and O blood-group antigens illustrate the importance of specific
glycosyltransferases.
These antigens, which can trigger harmful immune reactions, are oligosaccharides present on
both glycoproteins and glycolipids on the surface of erythrocytes
Blood type O: sticking out of rbc is: glucose, 2 galactose, n acetylglucosamine, and fucose
Made by: Dalia Aljayar
Edited by: Basel jobair

The A antigen: blood type O with addition of N-acetylgalactosamine attached to the outer
galactose residue
the B antigen: blood type O with addition of an extra galactose residue attached to the outer
galactose.
AB antigen: Blood type O with addition of both galactose residue and N-acetylgalactosamine

Carbs determines blood group

How enzymes end in the lysosome Mannose 6-P Targets Proteins to Lysosomes
1. Vesicles containing the M6P receptor and bound lysosomal enzyme bud from the trans-
Golgi network and then fuse with a sorting vesicle i.e. the late endosome.
protein is made in the ER and then sent to CIS Golgi
Mannose 6 phosphate in Golgi, Phosphorylate the mannose
As protein goes through Golgi, mannose helps enzyme to attach to receptors on the inside of
the Golgi
Attaching to a receptor, causes a vesicle to form that goes to lysosome. a clathrin coating forms
when the attachment of a protein to its receptor occurs.
Along the way clathrin is lost

FYI: Clathrins are important for protein selection and for specifying the destination. The
formation of a clathrin coat is important to the mechanism of vesicle budding as well as
receptor sorting.
2. The endosome (vesicle) has an internal pH of ≈5.5. This causes M6P bound lysosomal
enzymes (proteins) to be released from the M6P receptor within the late endosomes.
Since it is acidic inside the late endosome it causes the protein to pull away from the receptor
3. Furthermore, a phosphatase within late endosomes removes the phosphate from
lysosomal enzymes, preventing their rebinding to the M6P receptor.
Two types of vesicles bud from late endosomes. One type contains lysosomal enzymes, they
fuse with lysosomes delivering the lysosomal enzyme to their destination. The other type of
vesicle recycles the M6P receptor back to the trans-Golgi network.
The receptor becomes a new vesicle that heads toward Golgi
Made by: Dalia Aljayar
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Clinical- I cell disease

A group of genetic disorders, termed lysosomal storage diseases, are due to the absence of one
or more lysosomal enzymes. As a result, undigested glycolipids, and extracellular components
(proteoglycans) that would normally be degraded by lysosomal enzymes accumulate in
lysosomes as large inclusions.
No enzyme? accumulate
I-cell disease is a particularly severe type of lysosomal storage disease in which multiple
enzymes are missing from the lysosomes.
people who can’t put phosphate on mannose get I cell disease “cause M-6-P is required in
making lysosomal proteins”
Cells from affected individuals lack the GlcNAc (n acetylglucosamine) phosphotransferase that
is required for formation of M6P residues on lysosomal enzymes in the cis-Golgi.

So the protein does not end up in the lysosome

Biochemical comparison of lysosomal enzymes from normal individuals with those from
patients with I-cell disease led to the initial discovery of mannose 6-phosphate as the lysosomal
sorting signal.

Lacking the M6P sorting signal, the lysosomal enzymes in I-cell patients are secreted rather than
sequestered (isolated) in lysosomes.

Import of proteins to the peroxisome

Peroxisome: single membrane organelle with all their proteins encoded in the nucleus.
Proteins enter the peroxisomes through selective import from the cytosol – post-translational.
Signal sequence for import proteins is sequence of three amino acids (ser-lys-leu) at C-terminal
end of polypeptide.
Import process may involve soluble receptor proteins, docking proteins on the peroxisome
membrane called peroxins.
Import receptor peroxin Pex5 accompanies protein cargo into the peroxisome.

Clinical -Zellweger Syndrome

Zellweger Syndrome: Mutation of the Pex2 peroxin, a peroxisomal integral membrane protein
required for protein transport
Individuals have ‘empty peroxisomes’ + severe abnormalities of brain, liver and kidney, and
they die soon after birth.
Made by: Dalia Aljayar
Edited by: Basel jobair

The role of SNARE’s in vesicle trafficking

SNARE proteins are made up of 60 members.
Function of SNARE proteins: is to mediate vesicle fusion, the fusion of vesicles with their target
membrane bound organelle ex: vesicle fusing with the plasma membrane.
Vesicles carry neurotransmitters

SNAREs also mediate docking of synaptic vesicles with the presynaptic membrane in neurons.
These SNAREs are the targets of the bacterial neurotoxins responsible botulism and tetanus.
The vesicular SNARE: synaptobrevin, forms a helical complex with The plasma membrane
SNAREs: syntaxin and SNAP-25. “names aren’t required”
Synaptotagmin, a vesicular Ca2+-binding protein, in the presence of Ca2+ causes the protein to
insert into the plasma membrane, catalyze membrane fusion, releasing the NeuroTransmitter.
Ca makes contractions stronger causing fusing of membrane, the neurotransmitter is then
released from the neuron
presynaptic and postsynaptic proteins have snares

Tetanus toxin and botulinum toxin (types B, D, F, and G) specifically cleave the vesicle SNARE
protein, synaptobrevin. Other botulinum toxins are proteases that cleave syntaxin and SNAP-25
found on the presynaptic plasma membrane.

• Botox affect snare, prevent release of NT “ex: ACH”, no contraction, no wrinkles

Secretion of Proteins from the Cell

Proteins not destined for cellular organelles are secreted from the cell.
Some proteins are released from the cell as the vesicle attaches to plasma membrane.
Other proteins are stored in cytoplasmic vesicles until stimulated to release their contents.

Constitutive Secretion: - continuous secretion

Vesicles carry the protein in a continuous process and release their contents to the exterior of
the cell.
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It operates for proteins regularly released from the cell, such as ECM proteins including
collagen, elastin, and fibronectin.

Regulated Secretion: - secreted only when needed

Proteins are released from cells only at certain times by a process of exocytosis.

These proteins generally play important regulatory roles.

Proteins are concentrated in the TGN and held in the cell in vesicles until an appropriate
stimulus is received for their release.
For example, insulin is released in response to elevated blood glucose.