19.

5 Protein Targeting and Sorting *Despite the existence of nuclear ribosomes, most
polypeptide synthesis occurs on cytoplasmic ribosomes
• A eukaryotic cell is likely to have billions of after mRNAs have been exported through the nuclear
proteins molecules, each representing at least pores
10,000 kinds of polypeptides
o Each polypeptide must find its way to the • Free ribosomes à where mRNAs associate
appropriate location within/outside the upon arriving in the cytoplasm
cell o Ribosomes not attached to any
• A limited number of these polypeptides are membrane
encoded by the genome of the mitochondrion • Shortly after translation begins, two main
o For plant cells, the chloroplast genome pathways for routing the newly forming
as well polypeptide products begin to diverge:
• Most are encoded by nuclear genes and are
synthesized by a process beginning in the cytosol 1. Cotranslational import
• Each of these polypeptides must then be directed • Utilized by ribosomes synthesizing
to its proper destination and must therefore have polypeptides destined for the
some sort of “postal code” ensuring its delivery to endomembrane system or for export from
the correct place the cell
• Ribosomes become attached to ER
The various compartments of eukaryotic cells can be membranes early in the translational
grouped into three categories: process, and the growing peptide chains are
1. The endomembrane system then transferred across (or inserted to, in the
• The interrelated system of membrane case of integral membrane proteins) the ER
compartments membrane as synthesis proceeds
• Includes: • Called as such because movement of the
i. Endoplasmic reticulum (ER) polypeptide across the ER or into the ER
ii. Golgi apparatus membrane is directly coupled to the
iii. Lysosomes translation process
iv. Secretory vesicles • The subsequent conveyance of such
v. Nuclear envelope proteins to their final destinations is carried
vi. Plasma membrane out by various membrane vesicles and the
2. Cytosol Golgi apparatus
3. Mitochondria, chloroplasts, peroxisomes (and
related organelles), and the interior of the nucleus 2. Posttranslational import
• Pathway for polypeptides destined for either
• Polypeptides encoded by nuclear genes are the cytosol or for the mitochondria,
routed to these compartments using several chloroplasts, peroxisomes, and the
different mechanisms nuclear interior
• The process begins with transcription of DNA à • Ribosomes synthesizing these types of
RNAs that are processed in the nucleus polypeptides remain free in the cytosol as
o Processed in the nucleus and then their final destination or are taken up by the
transported through nuclear pores for appropriate organelle
translation in the cytoplasm, where most • The uptake by organelles of such completed
ribosomes are found polypeptides requires the presence of
• Although translation is largely a cytoplasmic special targeting signals
process, some evidence suggests that up to 10% • In the case of the nucleus, polypeptides
of a cell’s ribosomes may actually reside in the enter through the nuclear pores
nucleus, where they can translate newly • Polypeptide entrance into mitochondria,
synthesized RNAs chloroplast, and peroxisomes involves a diff.
• Nuclear translation appears to function mainly as kind of mechanism
a quality control mechanism
o Checks new mRNAs for the presence of
errors
Cotranslational Import Allows Some Polypeptides to Signal Hypothesis
Enter the ER as They Are Being Synthesized • First suggested in 1971 by Günter Blobel and
David Sabatini
• Cotranslational import into the ER is the first step • Proposed that some sort of intrinsic molecular
in the pathway for delivering newly synthesized signal distinguishes such polypeptides from the
proteins to various locations within the many polypeptides destined to be released into
endomembrane system the cytosol
• Proteins handled in this way are synthesized on • Stated that for polypeptides destined for the ER,
ribosomes that become attached to the ER the first segment of the polypeptide to be
shortly after translation begins synthesized, the N-terminus, contains an ER
o The role of the ER in this process was signal sequence
first suggested by experiments in which o The ER signal sequence directs the
Colvin Redman and David Sabatini ribosome-mRNA-polypeptide complex to
studied protein synthesis in isolated the surface of the rough ER where the
vesicles of rough ER complex anchors at a protein “dock” on
§ These vesicles are known as the ER surface
microsomes à can be isolated o As the polypeptide chain elongates
using subcellular franctionation during mRNA translation, it progressively
and centrifugation crosses the ER membrane and enters
o After brief incubation of the rough ER the ER lumen
vesicles in the presence of radioactive
amino acids and other components Synthesis of the small subunit, or light chain, of the
needed for protein synthesis, they protein immunoglobin G.
stopped the reaction by adding
puromycin • Shortly after the signal hypothesis was first
§ Puromycin à an antibiotic that proposed, the evidence for the actual existence
causes partially completed of ER signal sequences was obtained by Cesar
polypeptide chains to be Milstein and his associates
released from ribosomes • In cell-free systems containing purified ribosomes
o When the ribosomes and membrane and the components required for protein
vesicles were separated and analyzed to synthesis, the mRNA encoding the immunoglobin
see where the newly made, radioactive light chain directs the synthesis of a polypeptide
polypeptide chains were located, a product that is 20 amino acids at its N-terminal
substantial fraction of the radioactivity end than the authentic light chain itself
was found inside the ER lumen • Adding microsomes (ER membranes) to this
o This suggests that newly forming system leads to the production of an
polypeptides pass into the lumen of the immunoglobin light chain of the correct size
ER as they are being synthesized • This suggests that the extra 20-amino acid
§ Allows them to be routed through segment is functioning as an ER signal sequence
the ER to their correct o This signal sequence is removed when
destinations the polypeptide moves into the ER
o In addition to their importance to human
health and in the molecular biology
laboratory, antibiotics have been
• Subsequent studies revealed that other
important tools for unraveling how
polypeptides destined for the ER also possess an
ribosomes function during translation
N-terminal sequence that is required for targeting
the protein to the ER and that is removed as the
If some polypeptides move directly into the lumen of the
polypeptide moves into the ER
ER as they are being synthesized, how does the cell
• Proteins containing such ER signal sequences at
determine which polypeptides are to be handled this way?
their N-terminus are referred to as preproteins
(e.g. prelysozyme, preproinsulin, and
pretrypsinogen)
ER signal sequences
• Typically 15-30 amino acids long
• Consist of three domains:
o Positively charged N-terminal region
§ May promote interaction with the
hydrophilic exterior of the ER
membrane
o Central hydrophobic region
§ May facilitate interaction of the
signal sequence with the
membrane’s lipid interior
o A polar region adjoining the site where
cleavage from the mature protein will
take place
• Only polypeptides with ER signal sequences can
be inserted into or across the ER membrane as
their synthesis proceeds

The Signal Recognition Particle (SRP) Attaches the

Ribosome-mRNA-Polypeptide Complex to the ER
Membrane
• Newly forming polypeptides must become
attached to the ER membrane before very much
of the polypeptide has emerged from the • The figure illustrates the role played by the SRP
ribosome in cotranslational import ^^^
o If translation were to continue without
attachment to the ER, the folding of the Mechanism:
growing polypeptide chain might bury the • mRNA encoding a polypeptide destined for the
signal sequence ER starts to be translated on a free ribosome
• Contrary to the original signal hypothesis, the ER • polypeptide synthesis proceeds until the ER
signal sequence does not itself initiate contact signal sequence has been formed and emerges
with the ER – the contact is mediated by a signal from the surface of the ribosome
recognition particle (SRP) o at this stage, SRP binds to the signal
o Recognizes and binds to the ER signal sequence and blocks further translation
sequence of the newly forming (1)
polypeptide and then binds to the ER • The SRP then binds the ribosome to a special
membrane structure in the ER membrane called a
o Consists of six different polypeptides translocon
complexed with a 300-nucleotide (7s) o Carries out the translocation of
molecule of RNA polypeptides across the ER membrane
o The protein components have three main o Translocon à a protein complex
active sites: composed of several components
§ One that recognizes and binds to involved in cotranslational import,
the ER signal sequence including:
§ One that interacts with the § An SRP receptor
ribosome to block further • To which SRP binds
translation § A ribosome receptor
§ One that binds to the ER • Holds ribosomes in
membrane place
§ Pore protein
• Forms a channel
through which the
growing polypeptide can
enter the ER lumen
 § Signal peptidase • If the hydrophobic segments fail to fold properly
• Enzyme that removed à BiP binds again to the polypeptide and the
the ER signal sequence cycle is repeated
• As seen in (2), SRP (bringing an attached • In this way, BiP uses energy from ATP hydrolysis
ribosome) first binds to the SRP receptor, to promote proper protein folding
allowing the ribosome to become attached to the • Folding is often accomplanied by the formation of
ribosome receptor disulfide bonds between cysteines located in
• GTP binds to both SRP and SRP receptor, different regions of a polypeptide chain
unblocking translation and causing transfer of the o Reaction is facilitated by protein
signal sequence to the pore protein disulfide isomerase
o The core of the pore is formed by three § Enzyme present in the ER lumen
subunits that form the Sec61 complex § Catalyzes the formation and
• Sec61 has a central channel that opens as the breakage of disulfide bonds
signal sequence is inserted (3) between cysteine residues
• GTP is then hydrolyzed, accompanied by release § Starts acting before the
of SRP (4) synthesis of a newly forming
• As the polypeptide elongates, it passes into the polypeptide has been completed
ER lumen, and signal peptidase cleaves the • Allows various disulfide
signal sequence, which is quickly degraded (5) bond combinations to be
• After polypeptide synthesis is completed, the final tested until the most
polypeptide is released into the ER lumen, the stable arrangement is
translocon channel is closed, and the ribosome found
detaches from the ER membrane and dissociates Proteins that repeatedly fail to fold properly can activate
into its subunits, releasing the mRNA (6) several types of quality control mechanisms…

Protein Folding and Quality Control Take Place Within Unfolded protein response (UPR)
the ER • Mechanism that uses sensor molecules in the ER
• After polypeptides are released into the ER membrane to detect misfolded proteins
lumen, they fold into their final shape and, in • These sensors activate signaling pathways that
some cases, assemble with other polypeptides to shut down the synthesis of most proteins while
form multisubunit proteins enhancing the production of those required for
o Facilated by molecular chaperones protein folding and degradation
• BiP or binding protein
o most abundant chaperone in the ER ER-associated degradation (ERAD)
lumen • Another type of quality control mechanism that
o member of the Hsp70 family of recognizes misfolded or unassembled proteins
chaperones • Exports or “retrotranslocates” them back across
o acts by binding to hydrophobic regions of the ER membrane to the cytosol, where they are
polypeptide chains, especially to regions degraded by proteasomes
enriched in the amino acids tryptophan,
phenylalanine, and leucine
• BiP revents aggregation by transiently binding to
the hydrophobic regions of unfolded polypeptides
as they emerge into the ER lumen
o stabilizes them and prevents them from
interacting with other unfolded
polypeptides
• Polypeptide chain is then released by BiP,
accompanied by ATP hydrolysis, giving the
polypeptide a brief opportunity to fold
• If polypeptide folds correctly à hydrophobic
regions become buried in the molecule’s interior
and can no longer bind to BiP
Proteins Released into the ER Lumen Are Routed to Stop-Transfer Sequences Mediate the Insertion of
the Golgi Apparatus, Secretory Vesicles, Lysosomes, Integral Membrane Proteins
or Back to the ER • Integral membrane proteins are typically
anchored to the lipid bilayer by one or more a-
Glycoproteins à make up most of the proteins helical transmembrane segments consisting of
synthesized on ribosomes attached to the ER 20-30 hydrophobic amino acids
• Proteins with covalently bound carbohydrate
groups How are such proteins retained within the ER membrane
• The initial glycosylation reactions that add these after synthesis rather than being released into the ER
carbohydrate chains take place in the ER (often lumen?
while the growing polypeptide is still being à There are two main mechanisms by which
synthesized) hydrophobic transmembrane segments anchor newly
• After polypeptides have been released into the forming polypeptide chains to the lipid bilayer of the ER
ER lumen, glycosylated, and folded, they are membrane:
delivered by various types of transport vesicles to
their destinations within the cell 1. Stop-Transfer Sequences
• The first stop in this transport pathway is the • Involves polypeptides with a typical ER signal
Golgi apparatus sequence at their N-terminus à allows an SRP to
o Where further glycosylation and bind the ribosome mRNA complex to the ER
processing of carbohydrate side chains membrane
may occur • Elongation of the polypeptide chain then
o The Golgi apparatus serves as a site for continues until the hydrophobic transmembrane
sorting and distributing proteins to other segment of the polypeptide is synthesized
locations • The stretch of amino acids functions as a stop-
transfer sequence
Soluble proteins o Halts translocation of the polypeptide
• The default pathway takes soluble proteins from through the ER membrane
the Golgi apparatus to secretory vesicles that • Translocation continues, but the rest of the
move to the cell surface and fuse with the plasma polypeptide chain remains on the cystolic side of
membrane the ER membrane
o Leads to the secretion of such proteins o Results in a transmembrane protein with
from the cell its N-terminus in the ER lumen and its C-
• Soluble proteins entering the Golgi apparatus that terminus in the cytosol
are not destined for secretion from the cell • Meanwhile, the hydrophobic stop-transfer signal
possess specific carbohydrate side chains and/or moves laterally out through a side opening in the
short amino acid signal sequences that target translocon and into the lipid bilayer, forming the
each protein to its appropriate location within the permanent transmembrane segment that
endomembrane system anchors the protein to the membrane
o Such proteins are often called type I
transmembrane proteins
A different signaling mechanism is used for proteins
whose final destination is the ER
• The C-terminus of these proteins usually contains
a KDEL sequence
o Consists of amino acids Lys-Asp-Glu-
Leu or a closely related sequence
• The Golgi apparatus employs a receptor protein
that brings to the KDEL sequence and delivers
the targeted protein back to the ER
2. Internal Start-Transfer Sequences
• Involves membrane proteins that lack a typical
signal sequence at their N-terminus, and instead
possess an internal start-transfer sequence
• Performs two functions:
o Acts as an ER signal sequence that
allows an SRP to bind the ribosome-
mRNA complex to the ER membrane
o Its hydrophobic region functions as a
membrane anchor that moves out
through a side opening in the translocon
and permanently embeds the
polypeptide in the lipid bilayer
• The orientation of the start-transfer sequence at
the time of insertion determines which terminus of
the polypeptide ends up in the ER lumen versus Mechanism:
the cytosol (1) The first transmembrane segment is inserted via
• Proteins whose C-terminus is in the ER lumen an internal start-transfer sequence
and N-terminus is in the cytosol are known as (2) A stop-transfer sequence halts insertion and
type II transmembrane proteins releases this first section of membrane spanning
amino acids laterally into the ER membrane
(3) Now the additional start- and stop-transfer
sequences come into play, as the next start-
transfer sequence is threaded into the translocon

• Threading continues until the next stop-transfer

sequence is encountered
o At this point the next membrane section
is released laterally into the ER
membrane
• This process continues until all of the membrane-
spanning sections are inserted through the
membrane
• Once a newly formed polypeptide has been
incorporated into the ER membrane by one of the
preceding mechanisms, it can either:
o Remain in place to function as an ER
membrane protein, or
• Many transmembrane proteins have a single o Be transported to other components of
membrane-spanning region, but many others the endomembrane system
have more than one membrane-spanning region § i.e Golgi apparatus, lysosomes,
• Transmembrane proteins with multiple nuclear envelope, or plasma
membrane-spanning regions are formed in a membrane
manner similar to single-pass transmembrane • Transport is carried out by a series of membrane
proteins that use the start- and stop-transfer bussing and fusing events in which membrane
strategy vesicles pinch off from one compartment of the
o However, an alternating pattern of start- endomembrane system and fuse with another
transfer and stop-transfer sequences compartment
creates a polypeptide containing multiple • The net result of transport of transmembrane
transmembrane segments that pass proteins to the surface is that the regions of the
back and forth across the membrane proteome that were originally in the lumen or the
ER eventually come to lie outside the cell
o The regions of the protein that were in the
cytosol remain there
Posttranslational Import Is an Alternative Mechanism Posttranslational Import Across Two Membranes
for Import into the ER Lumen Allows Some Polypeptides to Enter Mitochondria and
Chloroplasts
à In contrast to the cotranslational import of proteins,
some proteins are synthesized in the cytosol and are • Proteins destined for the nuclear interior,
subsequently transported into the ER lumen mitochondria, chloroplasts, or peroxisomes are
imported into these organelles after translation
Posttranslational import has been completed – thus they represent
• Accomplished some of the same components as posttranslational import
cotranslational import, but other components • Since such proteins are synthesized on free
differ ribosomes and released into the cytosol, each
protein must carry a targeting signal that directs it
to the correct organelle
• Posttranslational import of proteins into the
nucleus depends on nuclear localization signals
that target proteins for transport through nuclear
pore complexes

Importing Polypeptides into Mitochondria and

Chloroplasts
• Although mitochondria and chloroplasts contain
their own DNA, they synthesize few of the
polypeptides they require
o More than 95% of the proteins residing in
these two organelles are encoded by
nuclear genes and synthesized on
cystolic ribosomes
o The small number of polypeptides
synthesized within mitochondria are
targeted mainly to their innter
mitochondrial membrane
o Polypeptides synthesized within the
Mechanism: chloroplasts are targeted mainly to
(1) As the polypeptide is synthesized, it associates thylakoid membranes
with chaperones of the Hsp70 family • Most mitochondrial and chloroplast polypeptides
o Keeps it unfolded so that it can pass are synthesized on cystolic ribosomes, released
through the ER membrane into the cytosol, and taken up by the appropriate
(2) A complex of ER membranes (called Sec62, 63, organelle (chloroplast/mitochondria) within a few
71, and 72) associated with sec61 pore targets minutes
the protein for translocation • The targeting signal for these polypeptides is
o The protein then moves through the pore, called a transit sequence
losing its associated chaperones as it o Located at the N-terminus of the
does so polypeptide
(3) The Sec protein complex associated with the o Once inside the mitochondrion or
pore also recruits the chaperone BiP to the site chloroplast, the transit sequence is
where the protein emerged into the ER lumen removed by a transit pepsidase located
o BiP then attaches to the polypeptide within the organelle
§ BiP is thought to couple ATP § Removal of transit sequence
hydrolysi to pulling the often occurs before transport is
polypeptide into the ER lumen, complete
using a ratchet-like, stepwise o Typically contain both hydrophobic and
mechanism hydrophilic amino acids
 • The uptake of polypeptide chains possessing
transit sequences is mediated by specialized
transport complexes located in the outer and
inner membranes of mitochondria and
chloroplasts

Mitochondrial transport complexes:

TOM à translocase of the outer mitochondrial membrane
TIM à translocase of the inner mitochondrial membrane

The comparable chloroplast complexes are:

TOC à translocase of the outer chloroplast membrane
TIC à translocase of the inner chloroplast membrane

• Polypeptides are initially selected for transport

into mitochondria or chloroplasts by components
of TOM or TOC known as transit sequence
receptors
• After a transit sequence has bound to its receptor,
the polypeptide containing this sequence is
translocated across the outer membrane through
a pore in the TOM/TOC complex
• If polypeptide is destined for organelle interior à
movement through the TOM/TOC complex is
quickly followed by passage through the TIM/TIC ^ a current model for the chaperone-mediated import of
complex of the inner membrane polypeptides intro the mitochondrial matrix
o Presumably at a contact side where the
outer and inner membranes lie close Mechanism:
together (1) Chaperones of Hsp70 class bind to a newly
forming polypeptide that is still in the process of
being synthesized in the cytosol, keeping it in a
• Polypeptides entering mitochondria and loosely folded state
chloroplasts must generally be in an unfolded (2) Transit sequence at the N-terminus of the
state before they can pass across membranes polypeptide binds to the receptor component of
bounding these organelles TOM, which protrudes from the surface of the
• To maintain this unfolded state, polypeptides outer mitochondrial membrane
targeted for mitochondria and chloroplasts are (3) Chaperone proteins are then released,
usually bound to chaperone proteins similar to accompanied by ATP hydrolysis, as the
those that help newly synthesized polypeptides polypeptide is translocated through the TOM and
fold correctly TIM pores and into the mitochondrial matrix
(4) When the transit sequence emerges into the
matrix, it is removed by transit peptidase
(5) As the rest of the polypeptide subsequently • For example, targeting a polypeptide to the outer
enters the matrix, mitochondrial Hsp70 molecules or inner mitochondrial membrane
bind to it temporarily. The subsequent release of o requires an N-terminal transit sequence to
Hsp70 requires ATP hydrolysis direct the polypeptide to the
• Thought to drive the translocation mitochondrion + and additional internal
process sequence called hydrophobic sorting
(6) Finally, in many cases, mitochondrial Hsp60 signal (to target polypeptide to its final
chaperone molecules bind to the polypeptide and destination)
help it achieve its fully folded conformation o in such cases, the hydrophobic sorting
signal acts as a stop-transfer sequence
that halts translocation of the polypeptide
• Both chloroplasts and mitochondria require through either the outer or inner
energy for the import of polypeptides membrane translocase
• Mitochondrial import is driven both by ATP § promotes its lateral movement
hydrolysis and by the electrochemical gradient out through a side opening in the
across the inner membrane translocase and into the lipid
• In contrast, chloroplasts maintain an bilayer of the membrane
electrochemical gradient across the thylakoid o the hydrophobic signal sequence then
membrane but not across the inner membrane remains embedded in the membrane,
o Presumably the energy requirement for anchoring the polypeptide to the lipid
import into the chloroplast stroma is met bilayer
by ATP alone § N-terminal transit sequence is
usually removed
• A combination of transit and hydrophobic sorting
Targeting Polypeptides to the Proper sequences is also used for targeting polypeptides
Compartments Within Mitochondria and to the intermembrane space
Chloroplasts o The polypeptide passes through the
outer membrane and the signal
• Due to the structural complexity of sequences are then removed, leaving the
mitochondria and chloroplasts, proteins to be polypeptide in the space between the two
imported from the cytosol must be targeted no membranes
only to the right organelle but also to the
appropriate compartment within the • In a similar fashion, multiple signals are involved
organelle in directing some chloroplast polypeptides to
• Mitochondria have four compartments: their final destination.
o Outer membrane
o Intermembrane space
o Inner membrane
o Matrix
• Chloroplasts have six compartments:
o Outer membrane
o Intermembrane space
o Inner membrane
o Stroma
o Thylakoid membrane
o Thylakoid lumen
à thus, the polypeptide may have to cross 1-3
membranes to reach its final destination

• Given the structural complexity of both organelles,

many mitochondrial and chloroplast polypeptides
require more than one signal to arrive at their
proper destinations