Download Membrane targeting of proteins

Chapter 3
Membrane targeting of proteins
By
D. Thomas Rutkowski & Vishwanath R. Lingappa
3.1 Introduction
• Cells must localize proteins to specific
organelles and membranes.
• Proteins are imported from the cytosol directly
into several types of organelles.
3.1 Introduction
• The endoplasmic reticulum (ER):
– is the entry point for proteins into the secretory pathway
– is highly specialized for that purpose
• Several other organelles and the plasma membrane receive
their proteins by way of the secretory pathway.
3.2 Proteins enter the secretory pathway by
translocation across the ER membrane (an
overview)
• Signal sequences target nascent secretory and
membrane proteins to the ER for translocation.
• Proteins cross the ER membrane through an
aqueous channel that is gated.
3.2 Proteins enter the secretory pathway by translocation across the ER membrane
• Secretory proteins translocate completely across the ER
membrane;
– transmembrane proteins are integrated into the membrane.
• Before leaving the ER, proteins are modified and folded by
enzymes and chaperones in the lumen.
3.3 Proteins use signal sequences to target
to the ER for translocation
• A protein targets to the ER via a signal
sequence, a short stretch of amino acids that
is usually at its amino terminus.
• The only feature common to all signal
sequences is a central, hydrophobic core that
is usually sufficient to translocate any
associated protein.
3.4 Signal sequences are recognized by the
signal recognition particle (SRP)
• SRP binds to signal sequences.
• Binding of SRP to the signal sequence slows
translation so that the nascent protein is
delivered to the ER still largely unsynthesized
and unfolded.
3.4 Signal sequences are recognized by the signal recognition particle (SRP)
• The structural flexibility of the M domain of
SRP54 allows SRP to recognize diverse
signal sequences.
3.5 An interaction between SRP and its
receptor allows proteins to dock at the ER
membrane
• Docking of SRP with its receptor brings the ribosome
and nascent chain into proximity with the translocon.
• Docking requires the GTP binding and hydrolysis
activities of SRP and its receptor.
3.6 The translocon is an aqueous channel
that conducts proteins
• Proteins translocate through an aqueous channel
composed of the Sec61 complex, located within the ER
membrane.
• Numerous accessory proteins that are involved in:
– Translocation
– Folding
– Modification associate with the channel
3.7 Translation is coupled to translocation
for most eukaryotic secretory and
transmembrane proteins
• An interaction between the translocon and the signal
sequence causes the channel to open and initiates
translocation.
• The exact mechanism of translocation may vary from one
protein to another.
3.8 Some proteins target and translocate
posttranslationally
• Posttranslational translocation proceeds
independently of both ribosomes and SRP.
• Posttranslational translocation is used
extensively in yeast but is less common in
higher eukaryotes.
3.8 Some proteins target and translocate posttranslationally
• The posttranslational translocon is distinct in
composition from the cotranslational
translocon, but they share the same channel.
3.9 ATP hydrolysis drives translocation
• The energy for posttranslational translocation comes
from ATP hydrolysis by the BiP protein within the ER
lumen.
• The energy source for cotranslational translocation is
less clear, but might be the same as for posttranslational
translocation.
3.9 ATP hydrolysis drives translocation
• Most translocation in bacteria occurs posttranslationally
through a channel that is evolutionarily related to the
Sec61 complex.
3.10 Transmembrane proteins move out of
the translocation channel and into the lipid
bilayer
• The synthesis of transmembrane proteins requires that
transmembrane domains be
– recognized
– integrated into the lipid bilayer
3.10 Transmembrane proteins move out of the translocation channel and into the lipid bilayer
• Transmembrane domains exit the translocon
by moving laterally through a protein-lipid
interface.
3.11 The orientation of transmembrane
proteins is determined as they are
integrated into the membrane
• Transmembrane domains must be oriented
with respect to the membrane.
• The mechanism of transmembrane domain
integration may vary considerably from one
protein to another
– especially for proteins that span the membrane
more than once
3.12 Signal sequences are removed by
signal peptidase
• Nascent chains are often subjected to
covalent modification in the ER lumen as they
translocate.
• The signal peptidase complex cleaves signal
sequences.
3.13 The lipid GPI is added to some
translocated proteins
• GPI addition covalently tethers the C-termini
of some proteins to the lipid bilayer.
3.14 Sugars are added to many
translocating proteins
• Oligosaccharyltransferase catalyzes N-linked glycosylation
on many proteins as they are translocated into the ER.
3.15 Chaperones assist folding of newly
translocated proteins
• Molecular chaperones associate with proteins in the
lumen and assist their folding.
3.16 Protein disulfide isomerase ensures
the formation of the correct disulfide bonds
as proteins fold
• Protein disulfide isomerases catalyze disulfide bond
formation and rearrangement in the ER.
3.17 The calnexin/calreticulin chaperoning
system recognizes carbohydrate
modifications
• Calnexin and calreticulin escort glycoproteins
through repeated cycles of chaperoning.
– The cycles are controlled by addition and removal of
glucose.
3.18 The assembly of proteins into
complexes is monitored
• Subunits that have not yet assembled into
complexes are retained in the ER by
interaction with chaperones.
3.19 Terminally misfolded proteins in the ER
are returned to the cytosol for degradation
• Translocated proteins can be exported to the
cytosol.
• There they are:
– ubiquitinated
– degraded by the proteasome
—a process known as ER-associated
degradation.
3.19 Terminally misfolded proteins in the ER are returned to the cytosol for degradation
• Proteins are returned to the cytosol by the
process of retrograde translocation.
– This is not as well understood as for translocation
into the ER.
3.20 Communication between the ER and
nucleus prevents the accumulation of
unfolded proteins in the lumen
• The unfolded protein response:
– monitors folding conditions in the ER lumen
– initiates a signaling pathway that increases the expression of genes
for ER chaperones
• The protein Ire1p mediates the unfolded protein response in
yeast by becoming activated in response to conditions of cellular
stress.
3.20 Communication between the ER and nucleus prevents the accumulation of unfolded proteins in the lumen
• Activated Ire1p splices HAC1 mRNA.
• It results in the production of the Hac1
protein, a transcription factor that:
– localizes to the nucleus
– binds to the promoters of genes with a UPR
response element
• The unfolded protein response in higher
eukaryotes has evolved more layers of
control beyond those seen in yeast.
3.21 The ER synthesizes the major cellular
phospholipids
• The major cellular phospholipids are synthesized
predominantly on the cytosolic face of the ER
membrane.
3.21 The ER synthesizes the major cellular phospholipids
• The localization of enzymes involved in lipid
biosynthesis can be controlled by the cell to
regulate the generation of new lipids.
• Cholesterol biosynthesis is regulated by
proteolysis of a transcription factor integrated
into the ER membrane.
3.22 Lipids must be moved from the ER to
the membranes of other organelles
• Each organelle has a unique composition of lipids.
– This requires that lipid transport from the ER to each
organelle be a specific process.
• The mechanisms of lipid transport between
organelles are unclear.
– They might involve direct contact between the ER and other
membranes in the cell.
• Transbilayer movement of lipids establishes
asymmetry of membrane leaflets.
3.23 The two leaflets of a membrane often
differ in lipid composition
• Movement of lipid molecules between the
leaflets of a bilayer is required to establish
asymmetry.
• Enzymes (“flippases”) are required for
movement of lipids between leaflets.
3.24 The ER is morphologically and
functionally subdivided
• The ER is morphologically subdivided into
specialized compartments, including:
– the rough ER for protein secretion
– the smooth ER for steroidogenesis and drug
detoxification
– the sarcoplasmic reticulum for calcium storage
and release
3.24 The ER is morphologically and functionally subdivided
• The functions of the smooth ER can be
specialized according to the needs of the
particular cell type.
• The ER may also be subdivided at the
molecular level, in ways not morphologically
evident.
3.25 The ER is a dynamic organelle
• The extent and composition of the ER change in
response to cellular need.
• The ER moves along the cytoskeleton.
3.25 The ER is a dynamic organelle
• The mechanisms by which the ER expands
and contracts and forms tubules have yet to
be discovered.
• The signaling pathways that control ER
composition are not yet understood but may
overlap with the unfolded protein response.
3.26 Signal sequences are also used to
target proteins to other organelles
• Signal sequences are used for targeting to and
translocation across the membranes of other
organelles.
• Mitochondria and chloroplasts are enclosed by a
double membrane, with each bilayer containing its
own type of translocon.
• Two distinct pathways target matrix proteins to
peroxisomes.
3.27 Import into mitochondria begins with
signal sequence recognition at the outer
membrane
• Mitochondria have an inner and an outer
membrane, each of which has a translocation
complex.
• Import into mitochondria is posttranslational.
3.27 Import into mitochondria begins with signal sequence recognition at the outer membrane
• Mitochondrial signal sequences are
recognized by a receptor at the outer
membrane.
3.28 Complexes in the inner and outer
membranes cooperate in mitochondrial
protein import
• The TOM and TIM complexes associate
physically, and the protein being imported
passes directly from one to the other.
• Hsp70 in the mitochondrial matrix and the
membrane potential across the inner
membrane provide the energy for import.
3.29 Proteins imported into chloroplasts
must also cross two membranes
• Import into chloroplasts occurs
posttranslationally.
• The inner and outer membranes have separate
translocation complexes that cooperate during
the import of proteins.
3.30 Proteins fold before they are imported
into peroxisomes
• Peroxisomal signal sequences are:
– recognized in the cytosol
– targeted to a translocation channel
• Peroxisomal proteins are imported after they are
folded.
3.30 Proteins fold before they are imported into peroxisomes
• The proteins that recognize peroxisomal
signal sequences remain bound during import
and cycle in and out of the organelle.
• Peroxisomal membranes originate by
budding from the ER.