INTRACELLULAR PROTEIN

TRANSPORT AND SORTING

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Signal for Protein Transport
 Newly synthesized protein must be transported
to the organelle where it functions.
 The transport is guided by sorting signals in its
amino acid sequence.
 Sorting signals are recognized by complementary
sorting receptors.
 The receptors function catalytically: after
completing one round of targeting, they return to
their point of origin to be reused.
 Most sorting receptors recognize classes of
proteins rather than an individual protein.

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Figure 12–6 A simplified of
protein traffic. Gated transport
(red), transmembrane transport
(blue), or vesicular transport
(green).
The sorting signals that direct a
a protein’s movement are
contained in each protein’s
amino acid sequence.
In principle, a sorting signal
could be required for either
retention in or exit from a
compartment.

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The Transport of Molecules Between
Nucleus and Cytosol

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The Transport of Molecules Between Nucleus and
Cytosol

The NPC
• Animal cells: ± 30 different NPC proteins or
nucleoporins.
• Nuclear envelope of a typical mammalian cell
contains 3000–4000 NPCs
• Total traffic passing each NPC: up to 500
macromolecules per second per NPC and can
transport in both directions at the same time.

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• NPC contains aqueous passages, through which
small water-soluble molecules can diffuse
passively.
• Small molecules (≤ 5000 daltons ) diffuse freely
• Large proteins traverse much more slowly;
• Proteins larger than 60,000 daltons can barely
enter by passive diffusion.
• NPC proteins that line the central pore contain
extensive unstructured regions forming a
disordered tangle, leaving small openings to allow
the diffusion of small molecules & blocking the
passage of large macromolecules.
 Bidirectional traffic occurs continuously between
the cytosol and the nucleus. 6
 Cytosolic ribosomes (30 nm in diameter ) cannot
diffuse through the NPC, confining protein
synthesis to the cytosol.
 How does nucleus exports large molecules (i.e.
ribosomal subunits) to the cytosol and import large
molecules. (i.e. DNA & RNA polymerases)?
 The molecules bind to specific receptor proteins
that ferry large molecules actively through
NPC.
 Proteins that function in the nucleus—i.e. histones,
DNA & RNA polymerases, gene regulatory
proteins, & RNA-processing proteins—are
selectively imported into the nucleus from the
cytosol. 7
• Bidirectional transport
• Import from cytosol to the nucleus
transported protein contains nuclear
localization signal
• Export from nucleus to cytosol
transported molecule contains nuclear
eksport signal

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Fungsi nuclear localization signal (NLS). Immunofluorescence micrographs
menunjukkan lokasi sel SV40 virus T-antigen yg memiliki nuclear localization
signal. (A) T-antigen protein normal mengandung sekuen kaya lysine diimport
ke nukleus (ditunjukkan oleh immunofluorescence yg distain dgn antibodi
terhadap T-antigen. (B) Mutasi T-antigen menyebabkan protein tetap berada
di sitosol.
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 Differences between NPC transport and organelle
membrane transpot:
NPC:
 Transport through large aquaeus pore
 Able to transport large perfectly folded
protein
Organelle membrane
 Through transporter protein in the lipid
bilayer
 Transported protein is not extensively folded.

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Transport of proteins into mitochondria
and chloroplasts

• Mitochondrion and chloroplast have their own DNA, but

most mitc and chlor proteins are encoded in nucleus
genome; therefore must be transported from the
• Transport mechanism is similar to both organelles:
• Unfolded protein is transported passing the outer and
inner membranes into the matrix space and stroma
• Involved chaperone proteinsTransport
• Protein transport passing several membranes is called
protein translocation

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• Two subcompartments in mitochondria:
• the internal matrix space
• the intermembrane space.
• Three subcompartments in chloroplasts:
• the internal matrix space
• the intermembrane space
• the thylakoid space surrounded by the
thylakoid membrane
• Proteins imported to mitochondrion and chloroplast
must be transported across a number of
membranes.
• The process of protein movement across
membranes is called protein translocation.
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Figure 12–21 The subcompartments
of mitochondria and chloroplasts.
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• Translocator Proteins in mitochondrion
membrane:
 TOM complex (translocase of the outer
mitochondrial membrane): transfer protein
across the outer membrane
 TIM complex (translocase of the inner
mitochondrial membrane). There are 2 TIM
complexes: TIM 23 & TIM 22: transfer
protein across the inner membrane
 SAM complex: helps proteins to fold properly in
the outer membrane.
 OXA complex: translocator in the inner
mitochondrial membrane, mediates
inner membrane proteins insertion.
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 TOM complex is needed to import mithocondrial
proteins encoded by nuclear DNA.
First TOM transports the protein signal sequent
into the intermembrane space and helps inserting
transmembrane proteins into the outer membrane
β-Barrell proteins (abundant in outer membrane),
are then passed on to the SAM complex.
 TIM23 complex transports soluble proteins into the
matrix space and helps to insert transmembrane
proteins into the inner membrane. TIM22 complex
mediates the insertion of a subclass of inner
membrane proteins, including the transporter that
moves ADP, ATP, and phosphate in and out of
mitochondria. 15
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• ATP hydrolysis and a membrane potential drive
protein import into the matrix space
• GTP & ATP hydrolysis drive translocation into
the chloroplast.
• Chaperon protein from the Hsp70 family in the
cytosol keep the protein precursor unfolded.
Hsp70 family in the matrix or stroma attract the
protein into organelles.
• Signal sequence:
• located at the N (amino) end can be cut after
import
• located in the middle can be maintained after
import.
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Figure 12–26 Role of energy in protein import into the mitochondrial
matrix space. (1) Bound cytosolic Hsp70 is released from protein (ATP
dependent). After insertion of signal sequence and of a portion of
polypeptide chain into TOM complex, signal sequence interacts with a
TIM complex. (2) The signal sequence is then translocated into matrix
space (requires a membrane potential across the inner membrane). (3)
Mitochondrial Hsp70 (part of an import ATPase complex), binds to
regions of polypeptide chain as they become exposed in the matrix
space, pulling the protein through the translocation channel.

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Signal sequent for protein import to mithocondrion

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 Two signal sequences direct proteins to the
thylakoid membrane in chloroplasts
 Both processes use separate translocation
complexes in each membrane, require energy, and
use amphiphilic N-terminal signal sequences that
are removed after use.
 Chloroplasts have an electrochemical H+ gradient
across their thylakoid membrane but not their inner
membrane.
 Energy needed for protein translocation into
chloroplast also comes from GTP & ATP hydrolysis.

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peroxisomes

• Single membrane organelles

• Plays a role in oxidative reaction,
• produces hydrogen peroxide used for oxidation
• contains catalase enzyme to destroy the excess
hydrogen peroxide
• The main function of oxidation in peroxisome is
breaking down fatty acid molecule to produce acetyl-
CoA.
• In mammalian cell ß-oxidation occurs in mitochondrion
and peroxisome
• In animal, peroxisome plays a role in initial reaction of
plasmalogen (phospholipids in myelin nerve) formation.
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 In plants, there are two types of peroxisomes:
1. In leaf, contribute to leaf photorespiration
2. In germinating seeds, convert fatty acid stored in the
grain into sugars required for growth of young plant.
 Conversion of fat into sugar is known as glyoxylate cycle;
therefore seed peroxyxome is called glioxysome.
 In glyoxylate cycle two moles of acetyl-CoA resulted from
the breakdown of fatty acid will be converted into glucose
in the cytosol.
 The glyoxylate cycle does not occur in animal cells;
therefore, animal cell is not capable of converting fatty acid
to carbohydrates.

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• Peroxisomes import all proteins from cytosol.
• Import Signal: three amino acids (Ser-Lys-Leu) at
the C terminal.
• Peroxin protein are responsible for protein
transport into peroxisomes (aboiut 23 types of
peroxin proteins).
• Large protein (oligomers) can be imported without
unfolding.

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Figure 12–38 The signal hypothesis.
Protein translocation across the ER membrane. When the ER signal
sequence emerges from the ribosome, it directs the ribosome to a
translocator on the ER membrane that forms a pore in the membrane
through which the polypeptide is translocated. A signal peptidase is
closely associated with the translocator and clips off the signal
sequence during translation, and the mature protein is released into
the lumen of the ER immediately after synthesis. The translocator is
closed until the ribosome has bound, so that the permeability barrier
of the ER membrane is maintained at all times. 24
• Pola glikosilasi N-linked indikasi seberapa
jauh pelipatan protein, protein meninggalkan RE
hanya setelah melipat sempurna.
• Protein yg tdk melipat & tdk mengalami
oligomerisasi dgn benar ditranslokasikan
kembali ke sitosol, dihilangkan glikosilasinya,
mengalami ubiquitilasi (ubiquitylated), &
didegradasi di proteasom.

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Figure 12–40 ER signal sequences and SRP direct ribosomes to the
ER membrane. <TTCC> The SRP and its receptor are thought to
act in concert. The SRP binds to both the exposed ER signal
sequence and the ribosome, thereby inducing a pause in
translation. The SRP receptor in the ER membrane, which is
composed of two different polypeptide chains, binds the SRP–
ribosome complex and directs it to the translocator. The SRP and
SRP receptor are then released, leaving the ribosome bound to
the translocator in the ER membrane. The translocator then
inserts the polypeptide chain into the membrane and transfers it
across the lipid bilayer.
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Figure 12–45. Soluble protein translocation across the ER membrane.
On binding an ER signal sequence (which acts as a start-transfer
signal), the translocator opens its pore, allowing the transfer of the
polypeptide chain across the lipid bilayer as a loop. After the protein
has been completely translocated, the pore closes, but the
translocator now opens laterally within the lipid bilayer, allowing the
hydrophobic signal sequence to diffuse into the bilayer, where it is
rapidly degraded. (In this figure, the ribosomes have been omitted
for clarity.)
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Figure 12–51 Protein glycosylation in the rough ER. Almost as soon as
a polypeptide chain enters the ER lumen, it is glycosylated on target
asparagine amino acids. The precursor oligosaccharide is transferred
to the asparagine as an intact unit in a reaction catalyzed by a
membrane-bound oligosaccharyl transferase enzyme. As with signal
peptidase, one copy of this enzyme is associated with each protein
translocator in the ER membrane. (The ribosome is not shown.)

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• Akumulasi protein dgn kesalahan pelipatan di
RE memicu unfolded protein response
aktivasi gen2 untuk mengatasi kondisi ini.
• Hanya protein2 yang membawa sekuen sinyal RE
akan diimport ke RE.
• Sekuen sinyal dikenali oleh partikel pengenalan
sinyal (signal recognition particle/SRP),
• SRP mengikat rantai polipeptida & ribosom;
juga mengarahkan ke protein reseptor di
permukaan sitosol membran RE kasar.

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• Pengikatan dgn membrane RE kasar
menginisiasi translokasi.
• Protein2 dpt larut yg ditujukan utk lumen RE,
utk sekresi atau ditransfer ke lumen organel2
lain secara sempurna memasuki lumen RE.
• Protein transmembran yg ditujukan utk RE atau
utk membrane sel lain sebagian ditranslokasikan
melintasi membrane RE, sebagian lagi tetap
terikat.

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