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Unit 6-1

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Unit 6-1

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Biol 266 – Cell Biology

UNIT 6
“Protein sorting to organelles - I”
A typical mammalian cell contains up to 10,000 different proteins,
and each must be localized to the correct organelle.

PROTEIN LOCATION
Na+/K+ ATPase plasma membrane
RNA polymerase nucleus
Proteases lysosome
Catalase peroxisome
ATP synthase mitochondria
Hormones extracellular space
Proteins synthesized in eukaryotic cells

A few proteins: Most of the proteins:


Are encoded by the DNA present Are encoded by nuclear DNA
in mitochondria and chloroplasts
Are synthesized on ribosomes in
Are synthesized on ribosomes inside the cytosol
mitochondria and chloroplasts
Are delivered to the organelle of
destination from the cytosol
Are incorporated directly into
compartments within mitochondria
and chloroplasts
Protein sorting: the process by which newly-made proteins are
directed to the correct location
Proteins A and B are sorted to different organelles

ribosome
mRNA

Protein A Protein B

organelle A organelle B
Each protein has a sorting signal (signal sequence) that can range
from 3-60 continuous amino acids. The signal sequence is often, but
not always, removed once the protein arrives at its destination.

ribosome
mRNA

Protein A Protein B

organelle A organelle B

signal sequence B
signal sequence A
Examples of signal sequences

_
COO

_
-Ser-Lys-Leu-COO
hydrophobic
positively charged
negatively charged
polar
Signal sequences are necessary and sufficient for protein sorting
3 steps in protein sorting

1. Recognition of the signal sequence by a shuttling cytosolic


receptor
2. Targeting to the outer surface of the organelle
membrane
3. Import of the targeted protein into the membrane or
transport of the protein across the membrane

A general problem for protein import into organelles:

How to transport the protein across membranes that are


normally impermeable to hydrophilic molecules
Three main mechanisms to import proteins into a membrane-enclosed organelle

Transport through
nuclear pores

Transport across
membranes

Transport by
vesicles
Three main mechanisms to import proteins into a membrane-enclosed organelle

Transport through
nuclear pores

Transports specific
proteins

Proteins remain folded


during transport
Three main mechanisms to import proteins into a membrane-enclosed organelle

Transport across
membranes

ER, mitochondria,
chloroplasts,
peroxisomes

Requires protein
translocators

Proteins are unfolded


in order to cross the
membrane
Three main mechanisms to import proteins into a membrane-enclosed organelle

Transport by
vesicles

From ER onward and


through
endomembrane system

Transport vesicles
collect cargo protein
and pinch off from
membrane

Deliver cargo by fusing


with another
compartment

Proteins remain folded


during transport
Nuclear import

Entry into the nucleus proceeds through a protein structure called the
nuclear pore complex (NPC). Composed of ~30 proteins, each with multiple
copies such that the NPC contains 500-1000 proteins when assembled.

Can transport 1000 molecules/sec, both directions simultaneously.

Small, water-soluble molecules and proteins up to ~40 kDa can move


through the nuclear pore complex (NPC) by passive diffusion.
Nuclear import

Project outwards and


helps channel cargo
Fibrils inside the
to the NPC
nucleus converge at
their distal ends to
form a basket,
function is not well
understood

anchor the NPC to


the nuclear envelope

line the central pore, membrane-bending,


many unstructured stabilize membrane
regions containing FG curvature
repeats, makes up
the meshlike nature
of the NPC
Nuclear import

Proteins synthesized in the cytoplasm are targeted for the nucleus by a


nuclear localization signal (NLS), e.g. P-K-K-K-R-K-V having basic residues.
1. Proteins with an NLS bind to an NLS receptor (importin a/b
heterodimer).
2. The protein/importin complex associates with cytoplasmic filaments.
3. The protein/importin complex passes through the NPC…..
4. …..and associates with a GTPase called Ran.
5. The Ran●GTP-importin b complex is transported back to the
cytoplasm where Ran is converted to Ran●GDP and brought back in to
the nucleus. Importin a is returned to the cytoplasm via a protein
called exportin.
The Ran “gradient” ensures directionality to nuclear transport.
The GTP-bound form only exists in the nucleus and the GDP-bound
form only exists in the cytosol.

“inactive”

“active”
Mitochondrial import

For import into the matrix, a (usually) N-terminal sorting sequence is


required. If the protein localizes to the intermembrane space, a
second sorting sequence is needed. Matrix-targeting sequences are
rich in hydrophobic, positively-charged and hydroxylated (Ser, Thr)
residues, but lack acidic residues. Tend to form amphipathic helix.

Import into the mitochondria only occurs at points where the inner
and outer membranes are in close contact.
1 Precursor proteins are kept in an
unfolded state by the action of
the cytosolic chaperone Hsc70.
This requires energy in the form
of ATP hydrolysis.

2 The matrix-targeting sequence


then interacts with a receptor in
the outer mitochondrial
membrane called TOM20 or
TOM22.

3 The receptor transfers the


protein to the general import pore
of the outer membrane composed
of the protein TOM40.

4,5 At contact sites between the


inner and outer membranes, the
protein passes through the import
pore of the inner membrane
composed of the proteins TIM23
and TIM17.
5 Matrix Hsc70 binds to TIM44.
ATP hydrolysis by this complex
helps power translocation of the
protein into the matrix.

6 As the matrix-targeting sequence


emerges in the matrix, it is
cleaved by a matrix protease.

7 The protein can then fold into its


final conformation, often (but not
always) assisted by matrix
chaperonins.

The H+ electrochemical gradient


generated by oxidative
phosphorylation is also required
for protein import into
mitochondria. This ensures that
only mitochondria that are
actively respiring can import
proteins. Hence, uncouplers block
import.
Targeting of proteins to the
intermembrane space requires a
second, hydrophobic targeting
sequence that does not allow the
protein to completely pass
through the TIM23/17 import
pore.

The stalled protein is then


released from the pore into the
membrane where a membrane-
anchored protease cuts the
protein, releasing it into the
intermembrane space.
ER import
Unlike the proteins that enter the nucleus, mitochondria,
chloroplasts and peroxisomes, most of the proteins that enter the
endoplasmic reticulum begin to be translocated (transported)
across the ER membrane before the protein is completely
synthesized.
This requires that the ribosome that is synthesizing the
protein be attached to the ER membrane, giving it a rough
appearance (and the name rough ER).

smooth ER rough ER
There are two separate populations of ribosomes in the cytosol:

1. membrane-bound ribosomes are attached to the cytosolic surface of


the ER membrane and are synthesizing proteins that are translocated
into the ER.
2. free ribosomes are unattached to any membrane and are synthesizing
all of the other proteins
The steps for importing a soluble protein into the ER lumen

1,2 The emerging polypeptide with its 3 SRP delivers the


ER signal sequence exposed is ribosome/polypeptide to the SRP
engaged by a complex of six receptor. This interaction is
proteins and an associated RNA enhanced by the binding of GTP to
molecule called the signal both SRP and its receptor.
recognition particle (SRP). This
binding halts translation and 4 The ribosome/polypeptide is then
delivers the ribosome/polypeptide transferred to the translocon,
to the ER. inducing it to open and receive the
polypeptide which enters as a
loop. Hydrolysis of GTP by SRP
and its receptor free these
factors for another round of
import.
5,6 Translation then resumes and the 7,8 Following completion of
signal sequence is cleaved by a translation, the ribosome is
membrane-bound protease called released causing the translocon to
signal peptidase. Following this close. The newly-synthesized
digestion, the rest of the protein protein then folds.
is synthesized and enters the
lumen of the ER.
Membrane proteins of the plasma membrane, Golgi, lysosome and
endosomes are all inserted into the ER membrane. From there, they are
transported to their correct location using amino acid and
carbohydrate sorting signals.

There are 6 main types of membrane-anchored proteins:


1. Type I: single pass, cleaved signal sequence at the N-terminus, uses
SRP-SRP receptor to get to ER membrane, Nout-Cin
2. Type II: single pass, no cleavable signal sequence, uses SRP-SRP
receptor to get to ER membrane, Nin-Cout
3. Type III: same as type II but Nout-Cin
4. Tail-anchored: single pass, no cleavable signal sequence, hydrophobic
membrane-spanning sequence at C-terminus, does not use SRP-SRP
receptor but the GET1/2/3 system to get to ER, posttranslational
insertion, Nin-Cout
5. Type IV: multispanning, no cleavable signal sequence, uses SRP-SRP
receptor for insertion of the first membrane-spanning domain but
not subsequent ones, IV-A are Nin-Cin, IV-B are Nout-Cin
6. GPI-anchored: entire protein is lumenal (out), cleaved signal
sequence at the N-terminus, uses SRP-SRP receptor to get to ER
membrane, anchored at C-terminus to membrane and then
transferred to GPI anchor.
(in=cytosol, out=lumen or extracellular space)
signal
yes no no no no yes
sequence*
SRP/SRP
yes yes yes no yes yes
receptor
* cleavable N-terminal signal sequence

cytosol (in)

lumen (out)

Type I Type II Type III tail- Type IV (B) GPI-


anchored anchored
Type I membrane proteins use a cleavable signal sequence and
a stop-transfer anchor (STA) sequence that acts as the
membrane spanning domain. The translocon opens to release
this hydrophobic stretch into the membrane.
Type II and III membrane proteins use a signal-anchor (SA) sequence
that acts as a dual signal sequence (directing the protein to the ER by
the SRP) and the anchor or membrane-spanning sequence.

The orientation is determined by the positioning of the SA sequence


within the translocon.

This is in turn determined by the positioning of positively-charged


residues relative to the SA sequence: if they are between the N-
terminus and the SA, then it will be Type II, if between SA and C-
terminus, then it will be Type III. These positive residues remain
cytosolic.

Type II Type III


Tail-anchored proteins are inserted into the ER after translation is
completed since the hydrophobic stretch that is inserted into the
bilayer needs to fully emerge from the ribosome.

1 The hydrophobic tail binds to


Get3 in its ATP-bound state.
Three other proteins (Get4/5 and
Sgt2) bind the hydrophobic tail
first before transfer to Get3.

2 The Get3-polypeptide complex


then binds to the Get1/2 complex
on the ER membrane.

3 Get3 then hydrolyzes ATP and


releases the polypeptide,
embedding its hydrophobic
stretch into the ER membrane.

4 Get3 binds ATP and is released


from the membrane, ready for
another round of activity.
GPI (glycosylphosphatidylinositol)-anchored proteins insert into the ER
like Type I membrane protein using a stop-transfer anchor (STA)
sequence. An enzyme (transamidase) then (i) cleaves the protein within
the lumen of the ER and (ii) transfers it to the assembled GPI anchor.

The purpose of transferring one lipid anchor for another:


1. The GPI anchor more readily diffuses in the membrane
2. GPI can act as a targeting signal (e.g. apical localization versus
basolateral).
Type IV membrane proteins use combinations of stop-transfer anchor
(STA) and signal-anchor (SA) sequences.

If the first SA sequence is a Type II SA (i.e. N-term+++++SA), then the


protein will be Nin (like a Type II membrane protein).

If the first SA sequence is a Type III SA (i.e. SA+++++C-term), then


the protein will be Nout (like a Type III membrane protein).
Hydropathic plots can help determine the type of membrane protein.
• The more hydrophobic an amino acid is, the more positive the hydropathic
index.
• The more hydrophilic the amino acid, the more negative the hydropathic
index.
The hydropathic index for groups of 20 amino acids are calculated and plotted
against the protein sequence.

Type I

Type IV

Type II or III (depends


on where the positive
charges are located
Besides proteins that reside in the ER, this organelle is the starting
point for:
1. Soluble proteins that will be secreted from the cell (e.g.
hormones)
2. Soluble proteins that are destined for the Golgi, lysosome or
endosomes (e.g. acid hydrolases)
3. Membrane proteins that will embed in the Golgi, lysosome,
endosomes or plasma membrane (e.g. Na+/K+-ATPase).

In order to export these proteins, the ER ensures that they are


properly modified, folded and assembled by a process known as
quality control.

Four principle modifications that occur in the ER:


1. Disulfide bond formation
2. Glycosylation (the addition and processing of carbohydrates)
3. Folding of polypeptides chains and assembly of multisubunit
complexes
4. Proteolytic cleavage of amino-terminal signal sequences
1. Disulfide bond formation – covalent bond formation between thiol groups
of cysteine residues either on the same protein (intramolecular) or on two
different proteins (intermolecular).

Disulfide bond formation is dependent upon the ER resident enzyme


protein disulfide isomerase (PDI). Thus, only (i) secreted proteins or
(ii) lumenal or extracellular domains of membrane proteins undergo this
modification.

Disulfide bonds stabilize protein structure – important for proteins


that will be subjected to either extremes in pH or environments with
high levels of proteases.
2. Glycosylation – begins with the addition of a common oligosaccharide
addition to asparagine residues in the consensus sequence Asn-X-Ser/Thr.
Referred to as N-linked glycosylation since the oligosaccharide is added to
the amine group of asparagine.

The precursor oligosaccharide is


transferred to the protein as the
consensus sequence emerges from the
translocon. Requires an ER membrane –
bound enzyme complex called
oligosaccharyl transferase.

The precursor oligosaccharide is


assembled in a step-wise fashion on a
lipid molecule called dolichol. N-acetylglucosamine
mannose
Trimming in the ER and Golgi usually glucose

does not involve these five sugar


residues and they can be thought of as
the core.
Dolichol contains 75-95 carbons. Assembly of the 2 N-acetylglucosamine
(GlcNAc) residues and the first 5 mannose residues takes place on the
cytosolic surface of the ER. The dolichol containing the seven sugar
residues then flips (using a transporter) to display the oligosaccharide in
the lumen of the ER. The remaining mannose residues and glucose
residues are added one at a time until the complete precursor is made.

Attachment of sugars to dolichol is mediated by nucleotides (UDP-


GlcNAc, UDP-glucose, GDP-mannose).
Tunicamycin is a molecule that blocks the attachment of the first
GlcNAc residue to dolichol. This results in non-glycosylated proteins.
Since glycosylation is used as a sign of protein folding, and folding is
required for export from the ER, this drug increases the level of
unfolded proteins in the ER inducing the unfolded protein response
(UPR).
The roles of glycosylation include:

1. Promote folding of proteins (e.g. protein secretion is


blocked for certain proteins when tunicamycin is used or if
Asn is mutated)

2. Provide stability to proteins (e.g. some non-glycosylated


proteins (fibronectin) are transported from the ER but are
degraded faster)

3. Promote cell-cell adhesion on plasma membrane proteins


(e.g. leukocyte-endothelial cell attachment during
inflammatory response)

4. Act as a transport signal (e.g. mannose-6-phosphate directs


proteins to the lysosome).
3. Folding – molecular chaperones assist in protein folding by preventing
aggregation of hydrophobic stretches of amino acids. Two types of ER
chaperones:
1. Classical chaperones (includes Hsp70 (BiP), Hsp90, GRP94)
2. Carbohydrate-binding chaperones (calnexin, calreticulin) – bind to
polypeptides that are monoglucosylated. Terminal glucose is removed
and if folded the protein can exit the ER. If not, a glucosyltransferase
adds one glucose back and the cycle repeats.

Ultimately, if mannose residues are removed, the protein is targeted for


dislocation (transport out of the ER) and degradation in the cytosol.

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