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Chapter 8
Protein
localization
8.1 Introduction
8.2 Chaperones may be required for protein folding
8.3 Post-translational membrane insertion depends on leader
sequences
8.4 A hierarchy of sequences determines location within
organelles
8.5 Signal sequences initiate translocation
8.6 How do proteins enter and leave membranes?
8.7 Anchor signals are needed for membrane residence
8.8 Bacteria use both co-translational and post-translational
translocation
8.9 Pores are used for nuclear ingress and egress
8.10 Protein degradation by proteasomes
8.1 Introduction
Leader of a protein is a short
N-terminal sequence
responsible for passage into or
through a membrane.
8.1 Introduction
Figure 8.1 Overview: proteins that are
localized post-translationally are
released into the cytosol after synthesis
on free ribosomes. Some have signals
for targeting to organelles such as the
nucleus or mitochondria. Proteins that
are localized cotranslationally associate
with the ER membrane during synthesis,
so their ribosomes are "membranebound". The proteins pass into the
endoplasmic reticulum, along to the
Golgi, and then through the plasma
membrane, unless they have signals that
cause retention at one of the steps on
the pathway. They may also be directed
to other organelles, such as endosomes
or lysosomes.
8.1 Introduction
Figure 8.2 Proteins synthesized on free ribosomes in the cytosol
are directed after their release to specific destinations by short
signal motifs.
8.1 Introduction
Figure 8.3 Membrane-bound
ribosomes have proteins
with N-terminal sequences
that enter the ER during
synthesis. The proteins may
flow through to the plasma
membrane or may be
diverted to other destinations
by specific signals.
8.2 Chaperones
may be required
for protein folding
Figure 8.4 A protein
is constrained to a
narrow passage as it
crosses a membrane.
8.2 Chaperones may be required for protein folding
Figure 8.5 Chaperone families have eukaryotic
and bacterial counterparts (named in parentheses).
8.3 The Hsp70 family is ubiquitous
Figure 8.6 DnaJ assists the binding of DnaK (Hsp70), which assists the folding of
nascent proteins. ATP hydrolysis drives conformational change. GrpE displaces the
ADP; this causes the chaperones to be released. Multiple cycles of association and
dissociation may occur during the folding of a substrate protein.
8.4 Hsp60/GroEL
forms an
oligomeric ring
structure
Figure 8.7-1 A
protein may be
sequestered within a
controlled
environment for
folding or
degradation.
8.4 Hsp60/GroEL
forms an
oligomeric ring
structure
Figure 8.7-2 GroEL
forms an oligomer of
two rings, each
comprising a hollow
cylinder made of 7
subunits.
8.4 Hsp60/GroEL
forms an oligomeric
ring structure
Figure 8.8 Two rings of
GroEL associate back to
back to form a hollow
cylinder. GroES forms a
dome that covers the
central cavity on one side.
Protein substrates bind to
the cavity in the distal ring.
8.4 Hsp60/GroEL
forms an oligomeric
ring structure
Figure 8.9 Protein
folding occurs in the
proximal GroEL ring
and requires ATP.
Release of substrate
and GroES requires
ATP hydrolysis in the
distal ring.
8.5 Post-translational
membrane insertion
depends on leader
sequences
Figure 8.10 Leader
sequences allow
proteins to recognize
mitochondrial or
chloroplast surfaces
by a post-translational
process.
8.5 Post-translational membrane insertion
depends on leader sequences
Figure 8.12 The leader sequence of yeast cytochrome c
oxidase subunit IV consists of 25 neutral and basic
amino acids. The first 12 amino acids are sufficient to
transport any attached polypeptide into the
mitochondrial matrix.
8.5 Post-translational membrane
insertion depends on leader sequences
Figure 8.13 TOM proteins form receptor complex(es)
that are needed for translocation across the
mitochondrial outer membrane.
8.5 Post-translational membrane
insertion depends on leader sequences
Figure 8.14 Tim proteins form the complex for translocation
across the mitochondrial inner membrane.
8.5 Post-translational
membrane insertion
depends on leader
sequences
Figure 8.15 Tim9-10
takes proteins from
TOM to either TIM
complex, and Tim813 takes proteins to
Tim22-54.
8.5 Post-translational
membrane insertion
depends on leader
sequences
Figure 8.16 A
translocating protein
may be transferred
directly from TOM
to Tim22-54.
8.6 A hierarchy of
sequences determines
location within
organelles
Figure 8.17 Mitochondria have receptors
for protein transport in the outer and
inner membranes. Recognition at the
outer membrane may lead to transport
through both receptors into the matrix,
where the leader is cleaved. If it has a
membrane-targeting signal, it may be reexported.
8.6 A hierarchy of sequences determines location
within organelles
Figure 8.18 The leader
of yeast cytochrome c1
contains an N-terminal
region that targets the
protein to the
mitochondrion, followed
by a region that targets
the (cleaved) protein to
the inner membrane. The
leader is removed by two
cleavage events.
8.6 A hierarchy of sequences determines location
within organelles
Figure 8.19 A protein
approaches the chloroplast
from the cytosol with a ~50
residue leader. The Nterminal half of the leader
sponsors passage into the
envelope or through it into
the stroma. Cleavage occurs
during envelope
8.7 Signal sequences initiate
translocation
Signal sequence is the region of a
protein (usually N-terminal)
responsible for co-translational
insertion into membranes of the
endoplasmic reticulum.
8.7 Signal sequences initiate translocation
Figure 8.20 The endoplasmic reticulum consists of a highly folded
sheet of membranes that extends from the nucleus. The small objects
attached to the outer surface of the membranes are ribosomes.
Photograph kindly provided by Lelio Orci.
8.7 Signal sequences initiate translocation
Figure 8.21 The signal sequence of bovine growth
hormone consists of the N-terminal 29 amino acids and
has a central highly hydrophobic region, preceded or
flanked by regions containing polar amino acids.
8.7 Signal sequences
initiate translocation
Figure 8.22 Ribosomes
synthesizing secretory
proteins are attached to
the membrane via the
signal sequence on the
nascent polypeptide.
8.7 Signal sequences initiate translocation
Figure 8.23 The two domains of the 7S RNA of the SRP are defined by its
relationship to the Alu sequence. Five of the six proteins bind directly to the
7S RNA. Each function of the SRP is associated with a particular protein(s).
8.8 The translocon
forms a pore
Figure 8.24 Does a
signal sequence
enter an aqueous
tunnel created by
resident ER
membrane proteins?
8.8 The
translocon forms
a pore
Figure 8.25 The
translocon consists
of SRP, SRP
receptor, Sec61,
TRAM, and signal
peptidase.
8.8 The
translocon forms
a pore
Figure 8.26 BiP
acts as a ratchet
to prevent
backward
diffusion of a
translocating
protein.
8.9 How do proteins enter and leave
membranes?
Integral membrane protein is a protein (noncovalently)
inserted into a membrane; it retains its membranous
association by means of a stretch of ~25 amino acids that
are uncharged and/or hydrophobic.
Transmembrane protein is a component of a membrane; a
hydrophobic region or regions of the protein resides in
the membrane, and hydrophilic regions are exposed on
one or both sides of the membrane.
8.9 How do proteins
enter and leave
membranes?
Figure 8.27 Group I
and group II
transmembrane
proteins have opposite
orientations with
regard to the
membrane.
8.9 How do proteins enter
and leave membranes?
Figure 8.28 The
orientations of the termini
of multiple membranespanning proteins depends
on whether there is an odd
or even number of
transmembrane segments.
8.9 How do proteins
enter and leave
membranes?
Figure 8.29-1 Does a signal
sequence interact directly
with the hydrophobic
environment of the lipid
bilayer or does it directly
enter an aqueous tunnel
created by resident ER
membrane proteins?
8.9 How do proteins
enter and leave
membranes?
Figure 8.29-2 How does a
transmembrane protein
make the transition from
moving through a
proteinaceous channel to
interacting directly with
the lipid bilayer?
8.9 How do proteins enter and leave membranes?
Figure 8.29-3
Proteins may
be associated
with one face
of a membrane
by acyl
linkages to
fatty acids.
8.10 Anchor signals
are needed for
membrane residence
Figure 8.30 Proteins that
reside in membranes enter by
the same route as secreted
proteins, but transfer is
halted when an anchor
sequence passes into the
membrane. If the anchor is at
the C-terminus, the bulk of
the protein passes through
the membrane and is exposed
on the far surface.
8.10 Anchor signals
are needed for
membrane residence
Figure 8.31 A combined
signal-anchor sequence
causes a protein to
reverse its orientation, so
that the N-terminus
remains on the inner
face and the C-terminus
is exposed on the outer
face of the membrane.
8.10 Anchor signals are needed for membrane residence
Figure 8.32 The signal-anchor of influenza neuraminidase is
located close to the N-terminus and has a hydrophobic core.
8. 11 Bacteria use both co-translational
and post-translational translocation
Figure 8.33 The Tat
and SecYEG ystems
are used for proteins
that are translocated
across the inner
membrane. YidC may
be used ith or without
SecYEG to insert
proteins into the inner
membrane.
8. 11 Bacteria use both co-translational
and post-translational translocation
Figure 8.34 SecB is a chaperone that transfers a nascent protein to SecA, which is a peripheral
membrane protein associated with the integral membrane protein complex SecYEG. Translocation
requires hydrolysis of ATP and a protonmotive force. Leader peptidase is an integral membrane
protein that cleaves the leader sequence.
8.12 Pores are used for nuclear ingress and egress
Figure 8.35 Nuclear pores are
used for import and export.
8.13 Nuclear pores
are large
symmetrical
structures
Figure 8.36 Nuclear
pores appear as
annular structures by
electron microscopy.
The bar is 0.5 mm.
Photograph kindly
provided by Ronald
Milligan.
8.13 Nuclear pores are
large symmetrical
structures
Figure 8.37 A model for the
nuclear pore shows 8-fold
symmetry. Two rings form
the upper and lower surfaces
(shown in yellow); they are
connected by the spokes
(shown in green on the inside
and blue on the outside).
Photograph kindly provided
by Ronald Milligan.
8.13 Nuclear pores are
large symmetrical
structures
Figure 8.38 The outsides of
the nuclear coaxial
(cytoplasmic and
nucleoplasmic) rings are
connected to radial arms. The
interior is connected to
spokes that project towards
the transporter that contains
the central pore.
8.13 Nuclear pores are large symmetrical structures
Figure 8.39 The
nuclear pore complex
spans the nuclear
envelope by means of a
triple ring structure.
The side view shows
two-fold symmetry
from either horizontal
or perpendicular axes.
8.13 Nuclear pores are large symmetrical structures
Figure 8.40 Nuclear localization signals have
basic residues.
8.15 Transport receptors carry cargo proteins through the pore
Exportins are transport receptors that bind cargo in the nucleus,
and translocate into the cytoplasm where they release the cargo.
Importins are transport receptors that bind cargo in the
cytoplasm, and translocate into the nucleus where they release
the cargo.
Nucleoporin was originally defined to describe the components
of the nuclear pore complex that bind to the inhibitory lectins,
but now is used to mean any component of the basic nuclear
pore complex.
Translocation of a chromosome describes a rearrangement in
which part of a chromosome is detached by breakage and then
becomes attached to some other chromosome.
8.15 Transport
receptors carry
cargo proteins
through the pore
Figure 8.42 There
are multiple
pathways for
nuclear export
and import.
8.15 Transport
receptors carry
cargo proteins
through the pore
Figure 8.41 A carrier
protein binds to a
substrate, moves
with it through the
nuclear pore, is
released on the other
side, and must be
returned for reuse.
8.15 Transport receptors carry cargo proteins through the pore
Figure 8.43 The assay for nuclear pore function
uses permeabilized cells.
8.15 Transport receptors
carry cargo proteins
through the pore
Figure 8.44 Nuclear
import takes place in two
stages. Both docking and
translocation depend on
cytosolic components.
Translocation requires
nucleoporins.
8.15 Transport
receptors carry
cargo proteins
through the pore
Figure 8.45 The state
of the guanine
nucleotide bound to
Ran controls
directionality of
nuclear import and
export.
8.15 Transport
receptors carry
cargo proteins
through the pore
Figure 8.46 Importin-b
consists of 19 HEAT
repeats organized in a righthanded superhelix. Each
HEAT unit consists of two
a-helices (A and B) lying at
an angle to one another.
Importin-b is folded tightly
around the IBB domain of
importin-a.
8.15 Transport
receptors carry
cargo proteins
through the pore
Figure 8.47 Importin-b
binds Ran-GDP through
close contacts to the Nterminal HEAT repeats
and to repeats 7-8. The
structure is significantly
different from the
importin-b importin-a
structure.
8.15 Transport
receptors carry
cargo proteins
through the pore
Figure 8.48 The
common feature in
proteins that are
exported from the
nucleus to the cytosol
is the presence of an
NES.
8.15 Transport
receptors carry
cargo proteins
through the pore
Figure 8.49 The ubiquitin
cycle involves three
activities. E1 is linked to
ubiquitin. E3 binds to the
substrate protein. E2
transfers ubiquitin from
E1 to the substrate.
Further cycles generate
polyubiquitin.
8.15 Transport
receptors carry
cargo proteins
through the pore
Figure 8.50 An archaeal
20S proteasome is a
hollow cylinder consisting
of rings of a and b
subunits. Photographs
kindly provided by Robert
Huber.
8.15 Transport
receptors carry
cargo proteins
through the pore
Figure 8.50 An archaeal
20S proteasome is a
hollow cylinder consisting
of rings of a and b
subunits. Photographs
kindly provided by Robert
Huber.
8.17 Summary
1. Synthesis of all proteins starts on ribosomes that are "free" in
the cytosol.
2. The N-terminal region of a secreted protein provides a signal
sequence that causes the nascent protein and its ribosome to
become attached to the membrane of the endoplasmic reticulum.
3. A secreted protein passes completely through the membrane
into the ER lumen.
4. Bacteria have components for membrane translocation that are
related to those of eukaryotes, but translocation often occurs by a
post-translational mechanism.
5. Nuclear pore complexes are massive structures embedded in the
nuclear membrane, and are responsible for all transport of protein
into the nucleus and RNA out of the nucleus.
6. Proteins that are actively transported into the nucleus require
specific NLS sequences, which are short, but do not seem to share
common features except for their basicity.
7. Proteins that are exported from the nucleus have specific NES
sequences, which share a pattern of leucine residues; they may
bind to nucleoporins.
8. The major system responsible for bulk degradation of proteins,
but also for certain specific processing events, is the proteasome, a
large complex that contains several protease activities.