Harvey Lodish • Arnold Berk • Paul Matsudaira •
Chris A. Kaiser • Monty Krieger • Matthew P. Scott •
Lawrence Zipursky • James Darnell
Molecular Cell Biology
Fifth Edition
Chapter 16:
Moving Proteins into Membranes
and Organelles
Copyright © 2004 by W. H. Freeman & Company
Protein sorting in different
organelles are governed by the same
basic mechanisms with variations
Questions for protein sorting
• Sequence context of the signal sequence
• Identity of the receptor for the signal
sequence
• Translocation channel structure and how
protein pass it
• Energy source for protein translocation
Signal sequence or Uptake-targeting
sequence
• Carrying the information for a protein to
target to a particular organelle destination.
• Signal sequences are often removed from
the mature protein by specific proteases.
Receptor proteins
• Each organelle carries a set of receptor
proteins that bind only to specific kinds of
signal sequences.
Translocation channel
• Translocation channel will allow the protein
to pass through the membrane once a
protein containing a signal sequence has
been recognized by its receptor protein.
Energy
• Protein translocation will only go
unidirectionally because it is energyconsuming.
Topics for this chapter
• ER protein targeting
• Bacterial protein export
• Targeting of proteins to mitochondria,
chloroplast, and peroxisomes
Translocation of secretory proteins
across the ER membrane
– In this book, secretory proteins are proteins that
will be secreted or will become luminal
proteins in the ER, Golgi, and lysosomes.
• Cotranslational translocation
• Post-translational translocation
Co-translational translocation of
secretory proteins across the ER
membrane
• Signal sequence: 16~30 residues with 6-12
hydrophobic residues (the core) and one or
more positively charged amino acids
adjacent to them. It will direct the ribosome
to the ER membrane and initiates
translocation of the growing peptide across
the ER membrane.
Co-translational translocation of
secretory proteins across the ER
membrane
• Receptor: SRP (signal-recognition particle)
and SRP receptor
– SRP is consisted of six proteins and a 300 nt
RNA
– SRP receptor is consisted of two subunits, a
and b (a is larger)
SRP (signal-recognition particle)
It use its hydrophobic binding
groove to interact with ER signal
sequence.
Co-translational translocation of
secretory proteins across the ER
membrane
• Translocation channel: Sec61 complex
– Sec61 is consisted of three subunit, a, b and g.
a is the largest, containing 10 membranespanning a helices.
• Energy source: GTP hydrolysis
Co-translational translocation of
secretory proteins across the ER
membrane
• Synthesis starts on
free ribosome first.
• The whole complex
must be translocated to
ER before the 70th
residue was added.
Without ER, the
synthesis will come to
a halt.
Co-translational translocation of
secretory proteins across the ER
membrane
• GTP hydrolysis is
required to increase
the fidelity of this
process. If the protein
does not have correct
signal sequence, GTP
hydrolysis will occur
and protein will leave
the complex.
Co-translational translocation of
secretory proteins across the ER
membrane
• If the protein is correct,
GTP hydrolysis will
trigger translocon
(Sec61) to open and
protein will enter ER
lumen.
Sec61 complex
• 5-6 nm (h) x 8.5 nm
(diameter)
• The pore is 2 nm in
diameter
Co-translational translocation of
secretory proteins across the ER
membrane
• By recognizing its Cterminal sequence,
signal peptidase will
cleave the ER signal
sequence after it enters
the ER lumen.
Co-translational translocation of
secretory proteins across the ER
membrane
• Signal sequence: 16-30 residues containing
6-12 residues of hydrophobic core with one
or more positively charged residues
adjacent to them
• Receptor: SRP and SRP receptor
• Translocon: Sec61 complex
• Energy requirement: GTP hydrolysis
Post-translational translocation of
secretory protein across ER
membrane
• Some yeast proteins are translocated into
ER by this pathway.
• Signal sequence context is the same as cotranslational translocation.
• Receptor: Sec63 complex and Bip
• Translocon: Sec61 complex
• Energy requirement: ATP hydrolysis
Post-translational translocation of
secretory protein across ER
membrane
• Translocating
polypeptide chain will
only slide back and
forth without Bip, a
member of Hsp70
family.
Post-translational translocation of
secretory protein across ER
membrane
• Bip hydrolyzes ATP
and Bip-ADP binds
translocating
polypeptide so it can
enter ER.
Post-translational translocation of
secretory protein across ER
membrane
• Bip-ADP pull
translocating
polypeptide chain into
ER.
Insertion of proteins into ER
membrane
• Main questions:
– How integral proteins can interact with
membranes?
– How topogenic sequences direct the insertion
and orientation of various classes of integral
proteins into the membrane?
• Integral membrane proteins
• GPI-anchored proteins
Topogenic sequences
• Membrane-spanning segments determine a
protein’s topology.
– 20-25 hydrophobic amino acids
– Form a-helix
Type I integral membrane proteins
• Single pass with
cleaved N-terminal
signal sequence
• C-terminal region on
the cytosolic side, Nterminal region on the
ER/Golgi lumen or
cell exterior
Type I integral membrane proteins
• Sequence context is the same as the signal sequence for ER
targeting
• Has stop-transfer anchor sequence
• Receptor: SRP, SRP receptor
Hydropathy profile
• Hydropathy profile can be used to search for
topogenic sequences.
Type II and III integral membrane
proteins
• Single pass with no
cleavable signal
sequence
• The orientations are
opposite
How type II and III membrane
proteins anchor differently?
• Positively charged
residues located on
one side of the
hydrophobic
sequences tend to stay
on the cytosolic side
that determines the
orientation of protein.
Type IV integral membrane proteins
• Multi-pass
• Can be further
categorized into IV-A
and IV-B, depending
on the orientation
Positively-charged segment
determines the orientation of integral
membrane proteins
Glycosylphosphatidylinositol (GPI)
GPI-anchored protein
• Some cell-surface proteins are anchored to
the phospholipid bilayer by a covalently
attached amphipathic molecule, GPI.
• Proteins with GPI anchors can diffuse in the
plane of the phospholipid bilayer membrane.
On the contrary, many proteins anchored by
membrane-spanning a helices are
immobilized in the membrane.
Synthesis of GPI-anchored proteins
• They are synthesized
and initially anchored
to the ER membrane
exactly like type I
transmembrane
proteins.
• A short sequence
adjacent to the
membrane-spanning
domain is recognized
by a transamidase.
Synthesis of GPI-anchored proteins
• Transamidase cleaves
off the original stoptransfer anchor
sequence and transfers
the remainder of the
protein to a preformed
GPI anchor in the
membrane.
Export of bacterial proteins
• Bacterial proteins will be exported to two
destinations:
– Inner membrane and periplasmic space
– Outer membrane
Export of bacterial proteins to the
inner membrane and periplasmic
space
• Signal sequence: N-terminal hydrophobic
sequence (will be cleaved by signal
peptidease later)
• Receptor: Ffh (similar to SRP) and FtsY
(similar to SRP receptor)
• Translocon: SecY, SecE and SecG; similar
to Sec61 complex
• Energy requirement: ATP hydrolysis
Inner membrane and periplasmic
space transport is post-translational
only
• SecA (similar to Bip) push the protein into
translocon.
Translocation across inner and outer
membrane to extracellular space
• Four types
– Type I and II: two-step translocation
• Substrate will be folded and acquire disulfide bonds
in the periplasmic space first
• Periplasmic proteins (spanning inner and outer
membranes) translocate folded proteins outside
• Energy requirement: ATP hydrolysis
Translocation across inner and outer
membrane to extracellular space
• Four types
– Type III and IV: single-step translocation
• Large protein complexes spanning both membranes
• Pathogenic bacteria use type III for protein secretion
and injection
• Agrobacterium tumefaciens (膿桿菌) use type IV to
transport T-DNA into plant cells.
Protein sorting to mitochondria and
chloroplast
• Mitochondrial protein targeting
– Mitochondrial matrix proteins
– Inner-membrane proteins
– Intermembrane space proteins
• Chloroplast protein targeting
– Stromal protein targeting
– Thylakoid protein targeting
Targeting for mitochondrial matrix
• Matrix-targeting sequence: 20-50 residues,
amphipathic helix with Arg and Lys
residues on one side and hydrophobic
residues on the other
• Receptor: import receptor
• Translocon: general import pore (Tom40,
Tim23/17 and Tim44, passive channel)
• Energy requirement: ATP hydrolysis
Hsc60
Mitochondrial inner membrane
protein targeting
• Energy comes from
proton motive force
• Three pathways:
– Path A use the same
machinery used for
targeting of matrix
proteins.
– Tom20/22 recognize their
matrix targeting sequence
Mitochondrial
inner membrane
protein targeting
• Precursors of path B
contain both a matrix
targeting sequence and
internal hydrophobic
domains recognized
by an inner-membrane
protein named Oxa1.
Mitochondrial
inner membrane
protein targeting
• Path C is used by
multi-pass proteins
containing multiple
internal mitochondrial
targeting sequences
recognized by Tom70.
Intermembrane-space proteins
targeting
Intermembrane-space protein
targeting
• Path A is similar to
pathway A for delivery
to the inner membrane,
but with
intermembrane spacetargeting sequence that
will be recognized by
an inner-membrane
protease.
Intermembrane-space protein
targeting
• Path B delivers protein
to intermembrane
space directly by
Tom40.
Outer-membrane protein targeting
• Sequence context: N-terminal short matrixtargeting sequence followed by a long stretch of
hydrophobic amino acids.
• Sequence will not be cleaved after translocation.
Chloroplast stromal protein targeting
• Sequence: no common motifs, generally
rich in Ser, Thr, and small hydrophobic
residues and poor in Glu and Asp.
• Import process is similar to mitochondria
matrix protein import but import proteins
are different.
• Chloroplast stroma does not have proton
motive force, so the energy of stromal
import comes solely from ATP hydrolysis.
Thylakoid protein targeting
• Four pathways
–
–
–
–
SRP-dependent pathway
SecA-related pathway
Oxa1 related protein pathway
ΔPH pathway
SRPdependent
pathway:
transport of
plastocyanin
(involved in
photophosphor
ylation)
to thylakoid
lumen
DPH pathway:
unfolded
proteins were
transported to
the stroma first,
then a set of
thylakoidmembrane
proteins will
transport them to
thylakoid lumen.
Thylakoidtargeting
sequence contain
two closely
spaced Arg.
Oxal related protein
pathway
• Some proteins are inserted
into the thylakoid
membrane by this pathway.
SecA-related protein pathway
• This pathway transport proteins into thylakoid
lumen.
Peroxisome protein targeting
• Sequence: PTS1 (SKL or related sequence)
near the C-terminus is required for
peroxisome targeting, it will not be cleaved
after transport
• ATP hydrolysis is required.
• Receptor: Pex5, Pex14
• Translocon: Pex10, Pex12
• Folded proteins can be translocated.
Principle modification for proteins
• Glycosylation: ER, Golgi
• Disulfide bond formation: ER
• Polypeptide chain folding and multisubunit
assembly: ER
• Proteolytic cleavage: ER, Golgi, secretory
vesicles
Glycosylation
• Glycosylation help folding, promote cellcell adhesion and confer stability.
• O-linked glycosylation can be found in
collagen and glycophorin, containing only
one to four sugar residues.
• N-linked glycosylation is more complex but
more common.
N-linked glycosylation
• The 14-residue oligosaccharide precursor for Nlinked glycosylation is synthesized in ER and will
be modified in ER and Golgi later.
The
oligosaccharide
precursor
• The three glucose
(glc) residues
serves as a signal
that this precursor
is complete and is
ready for transfer.
N-linked glycosylation
• The 14-residue precursor will be transferred from
dolichol to the asparagine residue of the target
protein with Asn-X-Ser or Asn-X-Thr sequence by
oligosaccharyl transferase.
• Then three glc and one man will be removed.
Disulfide bond formation
• In eukaryotic cells, disulfide bonds are
formed only in the lumen of the rough ER;
in bacterial cells, disulfide bonds are formed
in the periplasmic space between the inner
and outer membranes. Thus, disulfide bonds
are found only in secretory proteins and in
the exoplasmic domains of membrane
proteins.
Disulfide bond formation
• Protein disulfide isomerase (PDI) can
catalyzed the formation of disulfide bond
and can correct wrongly-formed disulfide
bond.
PDI will be oxidized by Ero1
• How Ero1
become
oxidized again
is not
clear….XD
Polypeptide chain folding
• Both BiP and PDI can help protein folding.
• Calnexin and calreticulin will bind
selectively to certain N-linked
oligosaccharides on growing nascent chains,
preventing aggregation of adjacent
segments of a protein as it is being made.
• Peptidyl-prolyl isomerases will accelerate
protein folding by rotating peptidyl-prolyl
bonds.
BiP and PDI help protein folding
Peptidyl-prolyl isomerase
• The rotation of peptidyl-prolyl bonds sometimes
are the rate-limiting step in the folding of protein
domains.
The unfolded
protein
response