16

Protein Targeting
The cells of eukaryotic organisms are architecturally intricate (Figure 1).
Goal To understand the molecular
mechanisms that are They have a nucleus in which the genetic material is stored and transcribed
responsible for the sorting of into RNA. They also have specialized organelles, such as mitochondria
proteins in eukaryotic cells. and, in the case of photosynthetic organisms, chloroplasts, which generate
chemical energy. Other organelles include lysosomes, which are sites of
Objectives degradation for macromolecules (a kind of eukaryotic trash can), and
After this chapter, you should be able to
peroxisomes, which house oxidative reactions that break down (catabolize)
fatty acids and other small molecules.
• explain how the architecture of
eukaryotic cells demands mechanisms The cytoplasm of eukaryotic cells also contains an endoplasmic reticulum
to target specific proteins to appropriate (ER), an extensive maze of interconnected spaces (lumena) surrounded by
destinations.
a membrane that serves as the site for the synthesis of proteins destined for
• describe how the decision to import into the ER. The ER is, in turn, connected to the Golgi apparatus,
enter either the cytoplasm or the a stack of flattened disks of membrane that receives proteins from the ER
endoplasmic reticulum is made on the
ribosome.
and directs them to other organelles, to the cytoplasmic membrane that
surrounds the cell, or to be secreted to the outside of the cell. The ER, the
• explain how the nuclear pore can block
Golgi, and all of the other individual organelles are surrounded by a single
the nuclear entry of some proteins but
not others. membrane bilayer, with the exception of the nucleus, mitochondria, and
chloroplasts. The nucleus is surrounded by a double membrane bilayer
• explain how proteins enter the
endoplasmic reticulum.
called the nuclear envelope that is contiguous with the endoplasmic
reticulum. Mitochondria and chloroplasts are also surrounded by inner
• explain the role of vesicles and coat
and outer membrane bilayers.
proteins in the secretory pathway.
• describe how v-SNAREs and t-SNAREs Thus, the ultrastructure of the eukaryotic cell is complex, posing challenging
enable vesicles to identify and fuse with questions about protein targeting. Each of the subcellular compartments
target membranes. of the cell contains unique proteins. How do these proteins get to their
Chapter 16 Protein Targeting 2

Figure 1 Proteins are targeted to

different cellular compartments
by a variety of mechanisms
The mechanism by which a protein
is trafficked to a particular cellular
compartment depends on the identity of transport across
the compartment. Proteins destined for chloroplast membranes
the nucleus are translated in the cytoplasm
and later translocated, in a folded state, nuclear mitochondrion
through nuclear pores. Proteins destined pore nucleus
for chloroplasts and mitochondria are
synthesized on ribosomes in the cytoplasm transport through
and later translocated into the target nuclear pores
compartment in an unfolded state. Proteins proteins
that are to be secreted and proteins that are
destined for the ER, Golgi, lysosome, or
cytoplasmic membrane are all translocated ER ribosomes
into the ER as they are synthesized. From
vesicle
the ER, these proteins travel in membrane-
bound vesicles to various cellular
compartments. transport by
Golgi vesicles

secreted protein

proper destination, be it the cytoplasmic membrane, the lysosome, the

mitochondrion, or the chloroplast? How do proteins become embedded in
membranes or cross membranes to reach a compartment on the other side
or for export out of the cell? Here we examine the ways in which the cell
addresses these protein sorting and transport challenges.

The first decision is whether to enter the endoplasmic reticulum

The journey of a protein to its proper destination is governed by a series of
decisions (Figure 2). The initial decision is whether to enter the endoplasmic
reticulum. ER entry is determined by the presence or absence of a short
stretch of amino acids at the N-terminus of a nascent polypeptide chain as
it emerges from the ribosome. These amino acids are known as the signal
sequence, and we will return to them presently. If a signal sequence is
absent, the newly synthesized protein is released into the cytoplasm. Such
proteins might remain in the cytoplasm (for example, many of the myriad
enzymes of the cell). However, particular amino-acid sequences (think of
them as addresses) target certain proteins to the nucleus, the mitochondria,
or other organelles.

Targeting the nucleus

Passage into (and out of) the nucleus occurs through specialized channels
known as nuclear pores (Figure 3). These protein-lined structures span
both the inner and outer membranes of the nuclear envelope. Nuclear
Chapter 16 Protein Targeting 3

The mRNA leaves the nucleus,

and its translation begins in the
cytoplasm.

Is there an ER signal sequence?

No Yes

SRP binds to the nascent ER

The translated protein is released
signal sequence, halting
into the cytoplasm.
translation until the ribosome
docks to the ER membrane.

Is there a
nuclear localization signal?
Is there a transmembrane
domain?
No Yes
No Yes
The translated protein is
The translated protein is released
Is there a mitochondrial embedded into the ER
The protein is trafficked to the into the ER lumen.
targeting sequence? membrane via its
nucleus.
transmembrane domain(s).

No Yes
Is there a second targeting Is there a second targeting
The protein is trafficked to the sequence? sequence?
The protein remains in the
cytoplasm. mitochondria.
No Yes Yes No

The protein is trafficked to the The protein is trafficked to the

cytoplasmic membrane and cytoplasmic membrane.
secreted.

The protein is retained in the The protein is retained in the

lumen of the targeted membrane of the targeted
compartment (e.g., ER, Golgi, compartment (e.g., ER, Golgi,
lysosome, etc.). lysosome, etc.).

Figure 2 Proteins are targeted to particular cellular locations depending on their targeting sequences
Shown is a decision tree that describes how particular sequence(s) affect protein-targeting decisions.

pores are large enough (100 nm) that ions and small molecules can freely
diffuse through them, but proteins cannot move through the pore without
assistance. This assistance takes the form of specialized proteins that act as
nuclear import receptors.

Figure 3 Proteins enter the cytoplasmic filament

nucleus through nuclear pores
central pore cytoplasmic ring
hydrogel outer nuclear membrane

nuclear envelope

inner nuclear membrane

nuclear basket nuclear ring
Chapter 16 Protein Targeting 4
The nuclear pore is lined with long, flexible, filamentous proteins that
interact with each other via backbone-backbone hydrogen bonding and
hydrophobic interactions to form a gel-like material known as a hydrogel.
Importantly, the hydrogel acts as a sieve, preventing large molecules from
passing through the channel. The import receptors recognize and bind to
proteins that bear a particular amino-acid sequence known as a nuclear
localization sequence. The nuclear import receptor, while bound to a
protein with a nuclear localization sequence, interacts with the hydrogel,
reshaping it so as to allow the protein complex to traverse the channel of the
nuclear pore and enter the nucleus.

Signal sequences target proteins to the ER

Now let’s consider the case of proteins that do have a signal sequence at
their N-termini (Figure 4). The signal sequence is generally a short stretch
of hydrophobic amino acids that targets proteins to the ER. The ER serves
as the entry point of proteins destined for other organelles. Proteins that are
targeted to the lysosome, the Golgi, and the cytoplasmic membrane all enter
the ER first. Once inside the ER, proteins do not re-enter the cytoplasm.
How does this targeting take place? Proteins that lack a signal sequence are
synthesized on ribosomes that are free and untethered in the cytoplasm.
However, proteins that contain a signal sequence are synthesized on
ribosomes that are associated with the outer surface of the ER. Indeed,
the ER can be covered with many such dot-like ribosomes, which impart
a rough appearance to its surface in electron micrographs (hence, it is
referred to as rough ER). The way this happens is as follows. Since the
signal sequence is at the N-terminus, when the mRNA for a protein that is
destined for the ER starts to be translated, the signal sequence is the first
part of the protein to emerge from the ribosome. The signal sequence in the
nascent protein is recognized by the signal recognition particle, which,
as we will see, attaches the ribosome to the ER membrane, resulting in a
membrane-attached ribosome. The ribosomal subunits, both large and
small, are recycled after each round of translation, and depending on which
mRNA they happen to translate, they will either become free or membrane-
attached. Thus, the two categories of ribosomes are functionally equivalent
and draw from a common pool of ribosomal subunits in the cytoplasm.
The signal recognition particle does two things (Figure 4). First, binding of
the signal recognition particle to the signal sequence of the nascent protein
temporarily slows translation. Second, the resulting complex, consisting
of the mRNA, ribosome, nascent protein chain, and signal recognition
particle, docks to a receptor on the surface of the ER known as the signal
recognition particle receptor. Once associated with the ER membrane, the
complex is directed to the protein translocation channel or translocon,
which is a transmembrane protein complex that spans the ER membrane and
provides a physical channel through which the unfolded, nascent peptide
can cross the ER membrane. Translation resumes at its normal rate once the
complex is docked with the translocon and the signal recognition particle
and signal recognition particle receptor dissociate from the complex. The
nascent peptide is then translocated through the channel as it is exported
from the ribosome in a process called co-translational translocation.
Chapter 16 Protein Targeting 5

Figure 4 Proteins enter the 2

secretory pathway via co- 1
5’
translational translocation, a small ribosomal
process mediated by the signal subunit 3’
recognition particle and other large ribosomal
protein factors subunit
mRNA nascent
Proteins that enter the secretory pathway 3’
peptide ER signal
bear a signal sequence that directs them
sequence
to the ER. The synthesis of such proteins
3 5’
initiates in the cytoplasm (State 1). As the
signal sequence of the nascent peptide
3’
emerges from the ribosome (State 2), it
is recognized by the signal recognition
particle (SRP in the figure), which binds to
both the signal sequence and the ribosome, signal recognition particle
slowing translation (State 3). The signal (SRP)
recognition particle carries the entire
ribosome-nascent chain complex to the ER 4 5’
membrane, where the signal recognition
particle binds to the signal recognition 5
3’ 5’
particle receptor (SRP receptor) in the ER
membrane (State 4). The entire complex is
then transferred to the translocon, at which 3’
time the SRP and SRP receptor dissociate,
allowing translation to resume (State 5). cytoplasm SRP receptor
Translation continues, and the nascent
protein is translocated in an unfolded ER membrane
state through the translocon and into the
ER lumen. The signal peptide is cleaved ER lumen
from the protein by a peptidase (State 6). translocon
When translation terminates, the ribosome
dissociates from the ER membrane and the
nascent protein is released into the ER lumen
5’
(State 7). Proteins with transmembrane
domains enter the secretory pathway via
the same mechanism, except instead of 6
7
being released into the ER lumen, their
transmembrane domains laterally exit the
translocon, moving sideways into the ER 3’
membrane (not shown).
cytoplasm

ER membrane

ER lumen

Proteins translated by ER membrane-attached ribosomes can be either soluble

(i.e., free-floating) proteins that are imported into the ER lumen (Figure 4)
or transmembrane proteins that are inserted into the ER membrane. Soluble
proteins simply cross the ER membrane during translation, after which
they are released into the ER lumen, where the signal sequence is removed
by a peptidase (signal peptidase). Transmembrane proteins are translocated
through the translocon until a transmembrane α-helix is reached. Instead
Chapter 16 Protein Targeting 6

Figure 5 Targeting sequences (A)

direct proteins to particular COO-
COO-
cellular compartments NH3+

Targeting sequences can either be signal

NH3+ sequence
continuous stretches of amino acids, as
in the case of the signal sequence (A) or (B)
discontinuous patches of amino acids that
COO-
come together when the protein folds (B). COO-
targeting
NH3+ sequence
NH3+ patch

of passing into the ER lumen, each transmembrane α-helix laterally exits

the translocon and enters the ER membrane. Transmembrane α-helices
contain stretches of hydrophobic amino acids that are recognized by the
translocon and trigger the lateral exit of the helix into the membrane.
Some proteins have additional targeting sequences, and in those cases the
sequences can function sequentially (Figure 5). For example, one sequence
could dictate entry into the ER, while a second, independent sequence can
dictate retention in the ER, preventing the protein from being sent on to
the cytoplasmic membrane, which might otherwise be its final destination
(Figure 6). As an example, imagine a secreted protein that is a dimer (i.e., a
protein whose folded structure consists of two polypeptide subunits). Here,
an ER retention signal could be used for “quality control” to ensure that only
correctly assembled dimers are secreted. In this case the ER retention signal
might be covered up by a second monomer once the protein assembles into
a correctly folded dimer. With the retention signal buried, the assembled
protein in its mature form can then proceed to the cytoplasmic membrane
for secretion from the cell. On the other hand, if the protein fails to assemble
properly, then the ER retention signal remains exposed, and the protein is
not trafficked to the cytoplasmic membrane, instead remaining in the ER.
The potassium leak channel, which we discussed in the previous chapter,
is an example of a protein that uses this quality-control mechanism. The

COO-

“enter the secretory pathway” COO-

COO- NH3+

NH3+ hidden retention

NH3+ signal
NH3+ “remain in the ER”

exposed retention signal COO-

Retained in ER Secreted from cell

Figure 6 Some proteins contain a retention sequence to ensure proper assembly
Shown is a protein with a signal sequence and a retention sequence. The blue signal sequence directs the protein into the lumen of
the ER, where the signal sequence is removed by cleavage. A second sequence, shown in red, causes the protein to be retained in the
ER, preventing it from progressing through the secretory pathway. When the protein forms a dimer, however, the ER retention signal
becomes concealed. When the retention signal is hidden, it is no longer recognized, allowing the protein dimer to progress through the
secretory pathway and be secreted from the cell.
Chapter 16 Protein Targeting 7
potassium channel is a tetramer (consisting of four polypeptide subunits).
The tetrameric structure is assembled in the ER, and an ER retention
signal is used to ensure that incorrectly assembled channels are not sent to
the cytoplasmic membrane. Each potassium channel monomer contains
a particular peptide sequence (Arg-Lys-Arg) that signals its retention in
the ER, but when the monomers assemble to form a tetramer, this peptide
sequence is covered up by other monomers, preventing it from being
recognized and thus allowing the assembled tetramer to exit the ER and be
trafficked to the cytoplasmic membrane. On the other hand, if the tetramer
fails to assemble correctly, the ER retention signal remains exposed and the
channel protein remains in the ER.

The secretory pathway

How do proteins migrate from the ER to the Golgi and from the Golgi to
other organelles, such as the lysosome and the cytoplasmic membrane, or
to be secreted to the outside of the cell? Broadly speaking, the targeting
of proteins to each of these organelles or to the extracellular space is
accomplished via a common series of steps known as the secretory pathway,
at the heart of which are small membrane vesicles (secretory vesicles)
(Figure 7). The vesicles that traffic proteins within the secretory pathway
are loaded with cargo proteins from the lumen of one compartment, and
they discharge their cargo into the lumen of a second compartment.
Secretory vesicles bud off from the membrane of one compartment and
fuse with the membrane of another. Vesicles carry cargo from the ER to the
Golgi and from the Golgi to various destinations, including endosomes,
lysosomes, and the cytoplasmic membrane. By default, proteins with only
an ER signal sequence will ultimately be trafficked to the cytoplasmic
membrane. An additional sequence is generally required for retention in

Figure 7 Proteins in the secretory cytoplasm extracellular

space
pathway are transported in vesicles nuclear envelope
between the ER, Golgi apparatus,
and cytoplasmic membrane ER
cytoplasmic
membrane

secretory
vesicle

Golgi apparatus
Chapter 16 Protein Targeting 8

cytoplasm 3
cytoplasmic
lumen membrane

transmembrane
protein donor compartment

vesicle

cytoplasm extracellular
space

Figure 8 Membrane protein topology is conserved during vesicular trafficking

The orientation of membrane proteins relative to the cytoplasm is conserved during vesicular transport. This figure shows an example of
a membrane protein that contains a region (colored red) that is exposed to the lumen of the donor compartment prior to transport to the
cytoplasmic membrane. As the secretory vesicle buds from the donor compartment membrane, the red region remains oriented towards
the interior of the vesicle (State 1). In the secretory vesicle this topology is conserved, and the red region of the protein remains on the
interior of the vesicle (State 2). When the vesicle fuses with the cytoplasmic membrane, the red region of the protein is exposed to the
extracellular space (State 3). At no point is the red region of the protein exposed to the cytoplasm. Similarly, any portion of a membrane
protein that is exposed to the cytoplasm is always exposed to the cytoplasm, even during transport.
the ER or Golgi or for trafficking to other compartments, such as lysosomes.
During the process of budding and fusion, the topology of the protein is
maintained (Figure 8). For example, if a transmembrane protein begins the
transport process with a domain in the lumen of the donor compartment,
that domain will face the lumen of the target compartment. If that
transmembrane protein is transported to the cytoplasmic membrane, the
domain that previously faced the lumen will face the extracellular space.
This also means that any part of a protein that is exposed to the cytoplasm
will remain exposed to the cytoplasm for the entire vesicular transport
process. The conservation of topology is important for the function of
many proteins.

Coat proteins promote budding and select protein cargo

The formation of secretory vesicles requires a protein coat that polymerizes
around the budding vesicle. The proteins coating a secretory vesicle play
critical roles in both physically forming the vesicle and in establishing cargo
selection. Vesicles bud off as coated vesicles that have a cage of proteins
covering their cytoplasmic surface. Three major types of coated vesicles,
named according to their protein coats, are clathrin, COPI (“cop one”),
and COPII (“cop two”). Each type participates in a particular route of the
transport process for a particular set of cargo molecules. For example,
COPII vesicles participate in the movement of cargo from the ER to the
Chapter 16 Protein Targeting 9
Golgi apparatus, whereas clathrin mediates the import of proteins from the
outside to the inside of the cell across the cytoplasmic membrane. Protein
coats have two major functions. First, they deform the membrane and
introduce curvature to form a vesicle. Second, they concentrate and select
for the appropriate cargo proteins.
The formation of a vesicle requires the membrane to curve, thereby
bringing negatively charged phospholipids into close proximity, an
energetically unfavorable state. The introduction of curvature is achieved in
part by neutralizing the negative charge on the phospholipid head groups,
making it more favorable for the membrane to be deformed. Curvature is
also introduced by the shape of the coat proteins themselves; some, like
clathrin, are known to have intrinsic curvature and to polymerize into a
partial sphere. The tight binding of the coat protein to the membrane and
the polymerization of the coat protein causes the membrane to deform.
Polymerization of coat proteins is a highly favorable process, and the energy
released during polymerization offsets the energetic cost of deforming the
membrane.
Coat proteins also select cargo. Coats accomplish this by interacting with
proteins that are to be enriched in the vesicle. These interactions may be
mediated through transmembrane proteins called adaptor proteins, which
connect the cargo inside the lumen of the vesicles with a particular protein
coat. The adaptor protein helps to specify which cargo molecules are
recruited by a particular protein coat. Adaptor proteins in turn recognize
and bind to specific sequences in cargo proteins.

SNARE proteins target vesicles to specific destinations and facilitate

membrane fusion
Vesicles must recognize their correct target membranes; because there
are so many membranes in the cell, a vesicle is likely to encounter many
Figure 9 SNAREs specify vesicle compartment 1
destinations and facilitate t-SNARE
membrane fusion
Coat proteins facilitate vesicle formation; coat protein
they also select particular cargoes and are
associated with specific v-SNAREs that v-SNARE
identify the vesicle origin. The v-SNAREs
in turn selectively bind to their cognate cargo
t-SNAREs on destination membranes. The
paired SNAREs facilitate membrane fusion, Budding Docking Fusion
and their specific interaction ensures
that cargoes are delivered to the correct
compartments.

v-SNARE

t-SNARE

compartment 2
Chapter 16 Protein Targeting 10
potential targets. Proteins called SNAREs determine which vesicles fuse
with which compartments (Figure 9). Specificity in targeting is ensured
because vesicles display v-SNAREs (vesicle SNAREs) on their surface that
identify them according to their origin and type of cargo; target membranes
display complementary t-SNAREs (target SNAREs) that recognize specific
v-SNAREs. The v-SNAREs are packaged together with the coat proteins
during the budding of transport vesicles. Contact with a target membrane
can trigger coat disassembly.
In addition to their role in targeting vesicles to the correct compartments,
SNAREs play a role in membrane fusion (Figure 10). Specifically, the
highly favorable interactions between cognate v- and t-SNAREs are used
to overcome the unfavorable electrostatic repulsion that occurs when two
membranes are brought together during fusion. v-SNAREs pair with their
cognate t-SNAREs to form a stable bundle of α-helices that forces the two
membranes into close apposition; the favorable energetics of the SNARE
pairing is used to overcome the unfavorable processes both of expelling
water molecules from the interface and of bringing charged phospholipid
head groups together. It is hypothesized that the phospholipids of the
two interacting leaflets then flow between the membranes to form a
connecting stalk. This rearrangement relieves the unfavorable head-to-
head interactions of the previous structure. Next, the lipids of the two other

1 v-SNAREs in the vesicle bind to 2 Water is squeezed from between the two 3 Stalk formation
complementary t-SNAREs in the membranes
target membrane

vesicle

v-SNARE
water

target t-SNARE
membrane

4 Hemi-fusion 5 Fusion

Figure 10 SNARE proteins allow vesicles to identify and fuse with specific target membranes
Chapter 16 Protein Targeting 11
leaflets contact each other, forming a new bilayer that widens the fusion
zone; this is known as the hemi-fusion state. Finally, rupture of the new
bilayer completes fusion of the vesicle and the target membrane.

Summary
Eukaryotic cells have an intricate architecture, with multiple membrane-
encased compartments. The presence of multiple membranes necessitates
mechanisms for delivering proteins to their proper destinations during or
after their release from the ribosome. A key decision point occurs on the
ribosome, when nascent proteins that display a signal sequence at their
N-terminus are targeted to the endoplasmic reticulum; those that lack a
signal sequence are released into the cytoplasm. Proteins that are released
into the cytoplasm can remain in the cytoplasm or travel to the nucleus
or to particular organelles, such as mitochondria and chloroplasts. For
example, proteins with a nuclear localization sequence are recognized
by nuclear import receptors, which escort them through channels in the
nucleus called nuclear pores. The pores are filled with filamentous proteins
that form a gel-like sieve that excludes proteins that are not in a complex
with a transport receptor.
Proteins that have a signal sequence at their N-terminus are recognized
by the signal recognition particle, which binds to the signal sequence as it
emerges from the ribosome and slows translation of the mRNA. The entire
complex of the arrested ribosome and the signal recognition particle then
binds to a receptor on the membrane of the ER, which in turn binds to
the translocon, a protein-conducting channel through which the nascent
protein is transported as it is translated. Soluble proteins are released
directly into the ER lumen, where their signal sequences are removed by
a peptidase. Membrane proteins laterally exit the translocon and enter the
ER membrane.
Transport of proteins among the ER, Golgi, cytoplasmic membrane, and
other compartments occurs via the secretory pathway. Proteins enter the
secretory pathway via the ER and are trafficked between the organelles of
the secretory pathway in secretory vesicles. Soluble proteins are trafficked
within the lumens of these vesicles, whereas transmembrane proteins are
embedded in the vesicle membrane. The default destination for proteins
in the secretory pathway is the cytoplasmic membrane, and additional
signaling sequences are typically needed for proteins to be retained in the
ER or Golgi or to be trafficked to other compartments.
Coat proteins have the dual function of facilitating vesicular budding and
selecting the cargo that enters each vesicle. For vesicles to bud, curvature
must be introduced into the membrane, which requires an input of energy.
Coat proteins interact favorably with one another and polymerize on
the outer surface of a budding vesicle. The energy released during this
polymerization is used to deform the membrane and promote budding.
Coat proteins also select cargo by binding to transport receptors that bind
to specific signal sequences in cargo proteins, enriching the vesicle in
appropriate cargo proteins.
Chapter 16 Protein Targeting 12
SNARE proteins ensure that vesicles fuse with the appropriate target
membranes. Each vesicle bears a particular set of SNARE proteins, called
v-SNAREs, which identify the origin of the vesicle. Each v-SNARE has a
complementary target SNARE (t-SNARE) on the target compartment. Each
particular v-SNARE pairs only with its cognate t-SNARE, ensuring that
vesicles only fuse to the appropriate target compartments. SNARE protein
pairing also facilitates membrane fusion. The v- and t-SNAREs form highly
favorable interactions with one another that draw the vesicular and target
membranes close together. The energy released as v- and t-SNAREs interact
is used to squeeze water molecules from between the two membranes and
to encourage the rearrangement and fusion of the membrane bilayers.