Moving proteins to membranes: Protein targeting and protein sorting in cells
Outer nuclear
membrane
Ribosomes
Inner nuclear
membrane
mRNA
1
Nucleus
Nuclear
pore
1
mRNA
Cytosol
ER signal
sequence
6
2
2
Cytosolic
protein
Rough endoplasmic
reticulum
Membrane
5
Matrix
Targeting
sequence
Peroxisome
4
Outer membrane
Intermembrane space
Matrix
3
Golgi
complex
3
Inner
membrane
Thylakoids
Stroma
Inner
membrane
Mitochondrion
Outer
membrane
Chloroplast
4a
Plasma
membrane
SECRETORY PATHWAY
4b
Lysosome
Ribosomes attached to the rough ER
Cytosol
Free ribosomes
ER lumen
Attached
ribosomes
ER membrane
0.5
m
Labeling experiments demonstrate that secretory proteins
are localized to the ER lumen shortly after synthesis
mRNA
Labeled
secretory
protein
Rough ER
Cells are incubated for a brief time with radiolabeled amino acids,
so that only newly synthesized proteins become labeled.
The cells then are homogenized, fracturing the plasma membrane
and shearing the rough ER into small vesicles called microsomes.
Because they have bound ribosomes, microsomeshave a much
greater density than other membranous organelles and can
be separated by sucrose density-gradient centrifugation
Homogenization
Microsomes
with attached
ribosomes
The purified microsomes are treated with a protease
in the presence or absence of a detergent.
Treat with
detergent
The labeled secretory proteins associated with the microsomes
are digested by added proteases only if the permeability barrier
of the microsomal membrane is first destroyed.
Add
protease
Digestion of
secretory protein
Add
protease
No digestion of
secretory protein
This finding indicates that the newly made proteins are inside the
microsomes, equivalent to the lumen of the rough ER.
Cell-free experiments demonstrate that translocation of
secretory proteins into microsomes is coupled to translation.
(a) Cell-free protein synthesis; no microsomes present
Add microsome
membranes
N-terminal
signal sequence
Completed proteins
with signal sequences
No incorporation
into microsomes;
no removal of
signal sequence
(b) Cell-free protein synthesis; microsomes present
Cotranslational transport
of protein into microsome
and removal of signal
sequence
Mature protein
chain without
signal sequence
Treatment of microsomes with EDTA,
which chelates Mg2+ ions, strips them
of associated ribosomes, allowing isolation
of ribosome-free microsomes, which are
equivalent to ER membranes
Synthesis is carried out in a cell-free
system containing functional ribosomes,
tRNAs, ATP, GTP, and cytosolic enzymes
to which mRNA encoding a secretory
protein is added. The secretory protein is
synthesized in the absence of microsomes
(a), but is translocated across the vesicle
membrane and loses its signal sequence
only if microsomes are present during
protein synthesis (b)
(a) Signal-recognition particle (SRP)
Structure of the signal-recognition
particle (SRP)
P19
P54
Binds ER
signal
sequence
P68/P72
RNA
Required for
protein
translocation
P9/P14
Interact with
ribosomes
(b) Ffh signal sequence–binding domain
(related to P54 subunit of SRP)
Hydrophobic
binding groove
(a) The SRP comprises one 300-nucleotide
RNA and six proteins designated P9, P14, P19,
P54, P68, and P72. (The numeral indicates the
molecular weight x 103.)
All proteins except P54 bind directly to the
RNA.
(b) The bacterial Ffh protein is homologous to
the portion of P54 that binds ER signal
sequences. This surface model shows the
binding domain in Ffh, which contains a large
cleft lined with hydrophobic amino acids
(purple) whose side chains interact with signal
sequences.
d
Synthesis of secretory proteins and their
cotranslational translocation across the ER membrane
SRP
mRNA
1
5'
2
NH 3 +
Signal
sequence
3
4
SRP receptor
GTP
Cytosol
!
ER
membrane
"
GDP + P i
3'
5
6
GDP + P i
7
GTP
ER lumen
8
Translocon
(closed)
Translocon
(open)
Signal
peptidase
Cleaved
signal
sequence
Folded
protein
Sec61α is a translocon component that contacts nascent
secretory proteins as they pass into the ER lumen
Artificial mRNA
5'
40S
subunit
40S
tRNA
60S
subunit
Ribosome
tRNA
60S
Cytosol
Sec61 !
Microsomal
membrane
Microsomal
lumen
Crosslinking
agent
Nascent
protein
10 nm
Translocon
NH 3 +
Cross-linking experiments show that Sec61α is a
translocon component that contacts nascent
secretory proteins as they pass into the ER lumen.
Electron microscopy
reconstruction reveals that a translocon
associates closely with a ribosome
Post-translational translocation across ER membrane
Translocating
polypeptide
chain
Translocon
Cytosol
ER lumen
Sec63
complex
1
NH 3 +
Cleaved
signal
sequence
ATP
BiP
(bound
to ATP)
Pi
ADP
2
ATP
Pi
ATP
ADP
3
Pi
ADP
4
ADP
ADP
ADP
ADP
ATP-Hydrolysis powers translocation
across the ER Membrane in yeast
ADP
5
ATP
ADP
ATP
6
ADP
Major topological classes of integral membrane
proteins synthesized on the rough ER
COO !
NH 3 +
COO !
Cytosol
Exoplasmic
space
(ER or Golgi
lumen;
cell exterior)
NH 3
+
Cleaved
signal
sequence
Type I
Glycophorin
LDL receptor
Influenza HA protein
Insulin receptor
Growth hormone
receptor
COO !
NH 3 +
Type III
COO !
Type II
Asialoglycoprotein receptor
Transferrin receptor
Sucrase–isomaltase precursor
Golgi galactosyltransferase
Golgi sialyltransferase
Influenza HN protein
Cytochrome P450
NH 3 +
Type IV
G protein–coupled receptors
(e.g., "-adrenergic receptor)
Glucose transporters (e.g., GLUT1)
Voltage-gated Ca 2+ channels
ABC small molecule pumps
!
CFTR (Cl ) channel
Sec61
Connexin
Synthesis and insertion into the ER membrane of
Type I single-pass proteins
Cytosol
5'
mRNA
1
2
4
3
5
COO !
Open
translocon
NH 3 +
Signal
peptidase
ER lumen
3'
6
Nascent
polypeptide
chain
Cleaved
signal
sequence
NH 3 +
Stop-transfer
anchor
sequence
NH 3 +
NH 3
+
NH 3 +
NH 3 +
Synthesis and insertion into the ER
membrane of Type II single-pass proteins
3
Nascent
polypeptide
chain
Cytosol
1
NH 3 +
2
5'
mRNA
NH 3 +
NH 3 +
3'
+
++
+
+
+
+
+
+
Translocon
ER lumen
Signalanchor
sequence
3
COO −
Arrangement of topogenic sequences in single-pass and multipass
membrane proteins inserted into the ER membrane
STA = Internal stop-transfer anchor sequence
SA-II = Internal signal-anchor sequence
SA-III = Internal signal-anchor sequence
(a) Type I
(b) Type II
(c) Type III
NH 3 +
NH 3 +
NH 3 +
COO !
Signal
sequence
STA
Lumen
Cytosol
+++
NH 3 +
NH 3 +
SA-II
+++
Cytosol
COO !
C y to s o l
Lumen
+++
Lumen
(e) Type IV-B
COO !
Cytosol
Lumen
SA-III
(d) Type IV-A
Cytosol
Lumen
Cytosol
++++++
S A -I I I
SA-II
+++
STA
SA-II
Cytosol
Lumen
S TA
+++
COO !
SA-II
STA
Cytosol
Lumen
SA-II
C y to s o l
Lumen
S TA
+++
Lumen
Cytosol
COO !
S A -I I
S TA
(a)
= Inositol
GPI-anchored proteins
= Glucosamine
= Mannose
PO 4
NH 2 = Phosphoethanolamine
PO 4
Fatty acyl chains
Hydrophobic
PO 4
NH 3 +
NH 3 +
Polar
(a) Structure of a glycosylphosphatidylinositol (GPI)
from yeast.
(b)
Cytosol
COO !
GPI
transamidase
NH 3 +
Preformed
GPI anchor
NH 3 +
Precursor
protein
ER lumen
(b) Formation of GPI-anchored proteins in the ER
membrane. The protein is synthesized and initially
inserted into the ER membrane. A specific
transamidase simultaneously cleaves the precursor
protein within the exoplasmic-facing domain, near
the stop-transfer anchor sequence (red), and
transfers the carboxyl group of the new Cterminus to the terminal amino group of a
preformed GPI anchor.
COO !
NH 3 +
NH 3 +
Mature GPI-linked
protein
Hydropathy profiles can identify likely topogenic
sequences in integral membrane proteins
(a) Human growth hormone receptor (type I)
4
3
2
1
0
!1
!2
!3
S igna l s equ ence
N-terminus
100
(b) Asialoglycoprotein receptor (type II)
4
3
2
1
0
!1
!2
!3
S i g n a l -a n c h o r s e q u e n c e
100
200
S to p - tr a n s f e r s e q u e n c e
200
300
400
500
C-terminus
(c) GLUT1 (type IV)
4
3
2
1
0
!1
!2
!3
T r a n s m e m br a n e s e qu e n ce s
100
200
300
400
Folding and assembly of hemagglutinin (HA) trimer in the ER
Oligosaccharyl
transferase
Dolichol
oligosaccharide
Calnexin
Cytosol
Membrane-spanning
! helix
Luminal
! helix
ER lumen
BiP
2
1a
PDI
S
S
3
1b
SH
HA 0 trimer
Calreticulin
Completed
HA 0 monomer
Post-translational translocation across
inner membrane in Gram-negative bacteria
SecA
ADP + P
ATP
Cytosol
ATP
i
ATP
1
ADP + P
ATP
i
ATP
2
1
ATP
2
1
2
Inner
membrane
Periplasmic
space
Translocon
(SecY, SecE, SecG)
The bacterial inner membrane contains a translocon channel composed of three subunits that are homologous to
the components of the eukaryotic Sec61 complex.
Translocation of polypeptides from the cytosol to the periplasmic space is powered by SecA, a cytosolic ATPase.
Binding and hydrolysis of ATP cause conformational changes in SecA, pushing the bound polypeptide segment
through the channel (steps 1, 2)
Repetition of this cycle results in movement of the polypeptide through the channel in one direction.
COO !
Protein import into
the mitochondrial matrix
Precursor
protein
ATP
ADP + P
i
1
Cytosolic
Hsc70
ATP
ADP + P
i
Precursor proteins synthesized on cytosolic
ribosomes are maintained in an unfolded or
partially folded state by bound chaperones, such as
Hsc70 (step 1).
Matrix-targeting
sequence
NH 3 +
Import
receptor
General
import pore
(Tom40)
2
3
Cytosol
4
Outer membrane
Tim23/17
Contact site
Tim44
Intermembrane
space
5
ATP
ADP + P
e
I n n e r m e m br a n
Matrix
Hsc70
i
Matrix
processing
protease
a trix
M it o ch o n dr ia l m
Active
protein
After a precursor protein binds to an import
receptor near a site of contact with the inner
membrane (step 2), it is transferred into the
general import pore (step 3).
6
7
Cleaved
targeting
sequence
The translocating protein then moves through this
channel and an adjacent channel in the inner
membrane (steps 4, 5).
Once the uptake-targeting sequence is removed by
a matrix protease and Hsc70 is released from the
newly imported protein (step 6), it folds into the
mature, active conformation within the matrix
(step 7).
Experiments with chimeric proteins show that a matrix-targeting
sequence alone directs proteins to the mitochondrial matrix and that
only unfolded proteins are translocated across both membranes.
(a)
COO
Unfolded
DHFR
!
(b)
Bound
methotrexate
inhibitor
COO
(c)
!
Outer
membrane
Intermembrane
space
Folded
DHFR
Cytosol
Outer membrane
Inner membrane
Intermembrane
space
In
r
ne
m
em
br
Mitochondrial
matrix
an
NH 3
e
+
Folded
DHFR
Cleaved
targeting
sequence
NH 3 +
Translocation
intermediate
Cleaved
targeting
sequence
Spacer sequence
Mitochondrial
matrix
0.2 m
Imported
protein
Location
of imported
protein
Locations of targeting sequences
in preprotein
Cleavage by
matrix protease
Alcohol
dehydrogenase III
Matrix
Matrix-targeting sequence
Cytochrome
oxidase
subunit
CoxVa
Inner
membrane
(path A)
Cleavage by
matrix protease
Cleavage by
matrix protease
ATP
synthase
subunit 9
Mature protein
Hydrophobic stoptransfer sequence
Internal sequences
recognized by Oxa1
Inner
membrane
(path B)
Internal sequences recognized
by Tom70 receptor and Tim22 complex
ADP/ATP
antiporter
Inner
membrane
(path C)
First cleavage by
matrix protease
Cytochrome
b2
Cytochrome
c heme lyase
Intermembrane
space
(path A)
Intermembrane
space
(path B)
Second cleavage by protease
in intermembrane space
Intermembrane
space–targeting sequence
Targeting sequence for
the general import pore
Stop-transfer and outermembrane localization sequence
Porin
(P70)
Outer
membrane
Arrangement of targeting
sequences in imported
mitochondrial proteins
Most mitochondrial proteins have an N-terminal
matrix-targeting sequence (pink) that is similar but
not identical in different proteins.
Proteins destined for the inner membrane, the
intermembrane space, or the outer membrane have
one or more additional targeting sequences that
function to direct the proteins to these locations by
several different pathways.
Three pathways for transporting proteins from the cytosol
to the inner mitochondrial membrane
P ath A
P ath B
Stop-transfer
sequence
Matrix-targeting
sequence
Preprotein
COO !
Cytosol
P ath C
Oxa1targeting
sequence
COO !
P r e p r o te i n
Tom22
Tom40
Internal targeting
sequences
Tom40
NH 3 +
P r o te i n
NH 3 +
NH 3 +
Tom20
COO !
Matrixtargeting
sequence
Tom40
Tom70
Outer
membrane
1
1
1
Intermembrane
space
Tim9/10
Tim23/17
Tim23/17
Tim22
Tim44
Inner
membrane
2
Tim54
2
Oxa1
Mitochondrial
matrix
Hsc70
Cleaved
matrixtargeting
sequences
Assembled
protein
Hsc70
3
2
NH 3 +
COO !
Two pathways for transporting proteins from the cytosol to the
mitochondrial intermembrane space
P ath A
P ath B
Intermembrane space–
targeting sequence
COO
Intermembrane space–
targeting sequence
Matrix-targeting
sequence
!
NH 3
Preprotein
Tom40
Tom20
Outer membrane
Intermembrane space
1
Heme
Tim44
3
2
ane
In ner m em br
Protease
Mitochondrial
matrix
Cleaved
matrix-targeting
sequence
NH 3 +
COO !
Tom22
Cytosol
Protein
+
Tim23/17
Tom40
Plastocyanin
COO ! precursor
Toc
complex
Metal-binding
precursor
COO
Thylakoid-targeting
sequence
Stromal-import
sequence
NH 3
NH 3 +
+
Toc
complex
Cytosol
Outer membrane
Intermembrane
space
Tic
complex
SRP-dependent
pathway
Metal-binding
protein
RR
Cleaved import
sequence
2
1
Tic
complex
Stroma
Plastocyanin
Chloroplast
SRP
Inner membrane
1
Cleaved import
sequence
2
Bound
metal
ions
RR
3
Thylakoid membrane
Chloroplast
SRP receptor
3
Thylakoid
lumen
4
RR
Mature
plastocyanin
"pH pathway
Mature
metal-binding
protein
!
Two of the four pathways for
transporting proteins from
the cytosol to the thylakoid
lumen.
Import of peroxisomal matrix proteins directed by
PTS1 targeting sequence
NH 3 +
COO
!
Step 1: Catalase and most other peroxisomal matrix
proteins contain a C-terminal PTS1 uptake-targeting
sequence (red) that binds to the cytosolic
receptor Pex5.
PTS1
peroxisomaltargeting sequence
1
Step 2: Pex5 with the bound matrix protein interacts
with the Pex14 receptor located on the peroxisome
membrane.
Pex5 receptor
Step 3: The matrix protein–Pex5 complex is then
transferred to a set of membrane proteins (Pex10,
Pex12, and Pex2) that are necessary for
translocation into the peroxisomal matrix by an
unknown mechanism.
2
4
Pex10
Cytosol
P erox is om
Pex14
a l m e m br a n e
Peroxisomal
matrix
Pex12
Pex2
3
Peroxisomal
matrix protein
Step 4: At some point, either during translocation or
in the lumen, Pex5 dissociates from the matrix
protein and returns to the cytosol, a process that
involves the Pex2/10/12 complex and additional
membrane and cytosolic
proteins not shown.