Download Protein Localization

Deuteronomy 6:5-9
5
And thou shalt love the LORD thy God with
all thine heart, and with all thy soul, and with
all thy might.
6
And these words, which I command thee this
day, shall be in thine heart:
7
And thou shalt teach them diligently unto thy
children, and shalt talk of them when thou
sittest in thine house, and when thou walkest
by the way, and when thou liest down, and
when thou risest up.
8
And thou shalt bind them for a sign upon thine
hand, and they shall be as frontlets between
thine eyes.
9
And thou shalt write them upon the posts of
thy house, and on thy gates.
©2001 Timothy G. Standish
Protein
Localization:
The Right Part in the Right Place
Timothy G. Standish, Ph. D.
©2001 Timothy G. Standish
DNA Does Not Specify Proteins
DNA sequence does not specify protein, but only the
amino acid sequence. The protein is one of a
number of minimum free-energy foldings of the
same amino acid chain, and the cellular milieu
together with the translation process influences
which of these foldings occurs … And organisms
are not determined by their DNA but by an
interaction of genes and the environment, modified
by random cellular events.
Lewontin, R. 2001 reviewing Who Wrote the Book of Life? A History of the Genetic
Code by Lily E. Kay. Science February 16, 2001.
©2001 Timothy G. Standish
Transcription And Translation
In Prokaryotes
5’
3’
3’
5’
RNA
Pol.
Ribosome
mRNA
Ribosome
5’
©2001 Timothy G. Standish
Eukaryotic Gene Expression
Cytoplasm
Packaging
Degradation
DNA
Transcription
Transportation
Modification
RNA
RNA
Processing
mRNA G
G
AAAAAA
Nucleus
Export
Degradation etc.
AAAAAA
Translation
©2001 Timothy G. Standish
After Translation
To be effective polypeptide chains
must:
1. Fold correctly - This may involve
chaperone protiens
2. Be modified, if necessary - for example,
by glycosylation at specific amino acids
3. Be in the correct location - Which can
be, as we shall see, a complex process
©2001 Timothy G. Standish
Protein Production and
Transport
Ribosomes
Cytoplasm
Rough
Nucleus
Endoplasmic
Reticulum
Smooth
Gogi
Complex
©2001 Timothy G. Standish
Protein Production
Mitochondria and Chloroplasts
Cytoplasm
Nucleus
G
AAAAAA
Export
Mitochondrion
Chloroplast
©2001 Timothy G. Standish
Protein Production
Mitochondria and Chloroplasts
Cytoplasm
Nucleus
Mitochondrion
Chloroplast
©2001 Timothy G. Standish
Protein Production
Mitochondria
Outer membrane
Inner membrane
Matrix
Inter membrane space
©2001 Timothy G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
ATP
ATP
P +ADP
Matrix
MLSLRQSIRFFKPATRTLCSSRYLL
P +ADP
Outer membrane
Inner membrane
Inter
membrane
space
©2001 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Peptidease
cleaves off
the leader
Inner membrane
Matrix
Inter
membrane
space
©2001 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Inter
membrane
space
©2001 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Inter
membrane
space
©2001 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Hsp60
Hsp60
Matrix
Chaperones
Inner membrane
Inter
membrane
space
©2001 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Mature protein
Inter
membrane
space
©2001 Timothy
G. Standish
M
S
L
R
Q
S
I
Yeast Cytochrome C
Oxidase Subunit IV Leader
L
First 12 residues are sufficient for
transport to the mitochondria
R
F
F
K
T
A
P
P
T
C
R
MLSLRQSIRFFKPATRTLCSSRYLL
R
L
S
S
Y
L
Neutral Non-polar
Polar
Basic
Acidic
This leader sequence probably forms an
a helix
This would localize specific classes of
amino acids in specific parts of the helix
There are about 3.6 amino acids per turn
of the helix with a rise of 0.54 nm per
turn
©2001 Timothy G. Standish
Yeast Cytochrome C1 Leader
Charged leader sequence signals
for transport to mitochondria
First cut
MFSNLSKRWAQRTLSKTLKGSKSAAGTATSYFEKLVTAGVAAAGITASTLLYANSLTAGA-------------Uncharged second leader sequence signals for transport
accross inner membrane into the intermembrane space
Second cut
Cytochrome c functions in electron transport and is
thus associated with the inner membrane on the
intermembrane space side
Cytochrome c1 holds an iron containing heme
group and is part of the B-C1 (III) complex
C1 accepts electrons from the Reiske protein and
passes them to cytochrome c
Neutral Non-polar
Polar
Basic
Acidic
©2001 Timothy G. Standish
Protein Production
Mitochondria
Outer membrane
Inner membrane
Matrix
Inter membrane space
©2001 Timothy G. Standish
Protein Production
Mitochondria
ATP
Leader sequence
binding receptor
P +ADP
Outer membrane
ATP
P +ADP
Peptidease
cleaves off
the leader
Matrix
Inner membrane
Inter
membrane
space
©2001 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Inter
membrane
space
©2001 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Inter
membrane
space
©2001 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Inter
membrane
space
©2001 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Inter
membrane
space
©2001 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Inter
membrane
space
©2001 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Inter
membrane
space
©2001 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Inter
membrane
space
©2001 Timothy
G. Standish
Protein Production
Mitochondria
Note that chaperones are not
involved in folding of proteins
in the inter membrane space
and that they exist in a low pH
environment
Leader sequence
binding receptor
Outer membrane
Inner membrane
Mature protein
Matrix
Inter
membrane
space
©2001 Timothy
G. Standish
Leader Sequence Receptors
Transporting proteins into the matrix actually involves two
receptors, one each for the outer and inner membranes:
1.TOM - A > 500 kD complex ~13.8 nm across composed of
~9 mostly transmembrane proteins in the outer membrane
• Tom40 Provides the channel for translocation
• Tom5,6,7 Are either assembly factors or part of the channel
• Tom20,22 Recognize most mtproteins via cytosol domains
• Tom37,70,71 Receptor for proteins with internal signal sequences
2.TIM - Two complexes on the inner membrane:
• Tim17-23 Recognizes signal sequence for translocation into the
matrix and probably provides the transmembrane channel
• Tim44 Binds both Tim17-23 on the matrix side of the inner
membrane and Hsp70 chaperone whose high affinity for unfolded
proteins helps to draw proteins in. Hsp 70 also binds another
chaperone, Mge.
©2001 Timothy G. Standish
Leader Sequence Receptors
Cytosol
Intermembrane
Matrix
T
O
M
37,
71,70
TIM44
Hsp70
T
T T
O
O O
M M M
40 5,6,722,
20
T
I
M
1723
Outer membrane
Inner membrane
MGE
©2001 Timothy G. Standish
Leader Sequence Receptors
Cytosol
Intermembrane
Matrix
T
O
M
37,
71,70
TIM44
Hsp70
T
T T
O
O O
M M M
40 5,6,722,
20
T
I
M
1723
Outer membrane
Inner membrane
MGE
©2001 Timothy G. Standish
Leader Sequence Receptors
Cytosol
Intermembrane
Matrix
T
O
M
37,
71,70
TIM44
Hsp70
T
T T
O
O O
M M M
40 5,6,722,
20
T
I
M
1723
Outer membrane
Inner membrane
MGE
©2001 Timothy G. Standish
Leader Sequence Receptors
Cytosol
Intermembrane
T
O
M
37,
71,70
TIM44
Hsp70
T
T T
O
O O
M M M
40 5,6,722,
20
T
I
M
1723
Outer membrane
Inner membrane
MGE
Matrix
©2001 Timothy G. Standish
Leader Sequence Receptors
Cytosol
Intermembrane
T
O
M
37,
71,70
TIM44
Hsp70
T
T T
O
O O
M M M
40 5,6,722,
20
T
I
M
1723
Outer membrane
Inner membrane
MGE
Matrix
©2001 Timothy G. Standish
Leader Sequence Receptors
Cytosol
Intermembrane
T
O
M
37,
71,70
TIM44
Hsp70
T
T T
O
O O
M M M
40 5,6,722,
20
T
I
M
1723
Outer membrane
Inner membrane
MGE
Matrix
©2001 Timothy G. Standish
Leader Sequence Receptors
Cytosol
Intermembrane
T
O
M
37,
71,70
TIM44
Hsp70
Matrix
T
T T
O
O O
M M M
40 5,6,722,
20
T
I
M
1723
Outer membrane
Inner membrane
MGE
Peptidease
©2001 Timothy G. Standish
Leader Sequence Receptors
Cytosol
Intermembrane
T
O
M
37,
71,70
TIM44
Hsp70
T
T T
O
O O
M M M
40 5,6,722,
20
T
I
M
1723
Outer membrane
Inner membrane
MGE
Matrix
©2001 Timothy G. Standish
Leader Sequence Receptors
Cytosol
Intermembrane
T
O
M
37,
71,70
TIM44
Hsp70
T
T T
O
O O
M M M
40 5,6,722,
20
T
I
M
1723
Outer membrane
Inner membrane
MGE
Matrix
©2001 Timothy G. Standish
Leader Sequence Receptors
Cytosol
Intermembrane
T
O
M
37,
71,70
TIM44
Hsp70
T
T T
O
O O
M M M
40 5,6,722,
20
T
I
M
1723
Outer membrane
Inner membrane
MGE
Matrix
©2001 Timothy G. Standish
Leader Sequence Receptors
Cytosol
Intermembrane
T
O
M
37,
71,70
TIM44
Hsp70
Matrix
T
T T
O
O O
M M M
40 5,6,722,
20
T
I
M
1723
Outer membrane
Inner membrane
MGE
Hsp60
©2001 Timothy G. Standish
Leader Sequence Receptors
Cytosol
Intermembrane
T
O
M
37,
71,70
TIM44
Hsp70
T
T T
O
O O
M M M
40 5,6,722,
20
T
I
M
1723
Outer membrane
Inner membrane
MGE
Matrix
©2001 Timothy G. Standish
Alternative Mechanism
1.
2.
There are actually two theories about how the
leader operates to localize mtproteins in the inter
membrane space:
The first, as shown in the previous slides,
involves the whole protein moving into and then
out of the matrix
The alternative theory suggests that once the first
leader, which targets to the mitochondria is
removed, the second leader prevents the protein
from ever entering the matrix so it is transported
only into the inter membrane space.
©2001 Timothy G. Standish
Leader Sequence Receptors
First part of leader signaling for
entrance into mitochondria
Cytosol
Intermembrane
Matrix
T
O
M
37,
71,70
TIM44
Hsp70
T
T T
O
O O
M M M
40 5,6,722,
20
T
I
M
1723
Outer membrane
Inner membrane
MGE
©2001 Timothy G. Standish
Leader
Sequence
Receptors
Second part of leader
First part of leader signaling for
entrance into mitochondria
signals for inter membrane space
Cytosol
Intermembrane
Matrix
T
O
M
37,
71,70
TIM44
Hsp70
T
T T
O
O O
M M M
40 5,6,722,
20
T
I
M
1723
Outer membrane
Inner membrane
MGE
©2001 Timothy G. Standish
Leader
Sequence
Receptors
Second part of leader
First part of leader signaling for
entrance into mitochondria
signals for inter membrane space
Cytosol
Intermembrane
T
O
M
37,
71,70
TIM44
Hsp70
T
T T
O
O O
M M M
40 5,6,722,
20
T
I
M
1723
Outer membrane
Inner membrane
MGE
Matrix
©2001 Timothy G. Standish
Leader
Sequence
Receptors
Second part of leader signals
for inter membrane space
Cytosol
Intermembrane
T
O
M
37,
71,70
TIM44
Hsp70
T
T T
Outer membrane
O
O O
M M M
40 5,6,722,
20
First part of leader signaling for entrance
T
into mitochondria
I
M
1723
Inner membrane
MGE
Matrix
©2001 Timothy G. Standish
Leader
Sequence
Receptors
Second part of leader signals
for inter membrane space
Cytosol
Intermembrane
T
O
M
37,
71,70
TIM44
Hsp70
T
T T
O
O O
M M M
40 5,6,722,
20
T
I
M
1723
MGE
Outer membrane
Inner membrane
First part of
leader signaling for
entrance into mitochondria
Matrix
©2001 Timothy G. Standish
Leader Sequence Receptors
Cytosol
Intermembrane
T
O
M
37,
71,70
Second part of leader signals
for inter membrane space
Matrix
TIM44
Hsp70
T
T T
O
O O
M M M
40 5,6,722,
20
Outer membrane
Second part of leader prevents
entrance into TIM 17-23
T
I
M
1723
MGE
Inner membrane
First part of leader signaling for
entrance into mitochondria
©2001 Timothy G. Standish
Leader Sequence Receptors
Cytosol
Intermembrane
Matrix
Second part of leader signals
for inter membrane space
T
O
M
37,
71,70
T
T T
O
O O
M M M
40 5,6,722,
20
Outer membrane
Second part of leader prevents
entrance into TIM 17-23
TIM44
Hsp70
T
I
M
1723
Inner membrane
First part of leader signaling
for entrance into
mitochondria
MGE
Peptidease
©2001 Timothy G. Standish
Leader Sequence Receptors
Cytosol
T
O
M
37,
71,70
Intermembrane
Peptidease
Matrix
Second part of leader signals
for inter membrane space
T
T T
O
O O
M M M
40 5,6,722,
20
Outer membrane
Polypeptide
passes
Second part of leader
prevents
through TOM, but
entrance into TIM 17-23
not TIM
TIM44
Hsp70
T
I
M
1723
MGE
Inner membrane
Peptidease
©2001 Timothy G. Standish
Leader Sequence Receptors
Cytosol
Intermembrane
Peptidease
Matrix
T
O
M
37,
71,70
T
T T
O
O O
M M M
40 5,6,722,
20
Outer membrane
Protein localized in the
intermembrane space
TIM44
Hsp70
T
I
M
1723
Inner membrane
MGE
©2001 Timothy G. Standish
1999 Nobel Prize in Physiology
and Medicine
Günter Blobel - For his
pioneering work in discovery
of signal sequences, the
molecular zip codes of protein
production and localization
©2001 Timothy G. Standish
©2001 Timothy G. Standish
The Genetic Code
Neutral Non-polar
Polar
Basic
Acidic
F
I U
R
S C
T
†Have amine
groups
*Listed as
non-polar by
some texts
B A
A
S G
E
SECOND
U
UUU
UUC
UUA
UUG
CUU
CUC
CUA
CUG
Phe
Leu
Leu
C
UCU
UCC
UCA
UCG
CCU
CCC
CCA
CCG
AUU
AUC Ile
AUA
AUGMet/start
ACU
ACC
ACA
ACG
GUU
GUC
GUA
GUG
GCU
GCC
GCA
GCG
Val
BASE
A
Ser
UAU
UAC
UAA
UAG
Tyr
Pro
CAU
CAC
CAA
CAG
His
Thr
AAU
AAC
AAA
AAG
Asn†
Ala
GAU
GAC
GAA
GAG
Asp
Stop
Gln†
Lys
Glu
G
UGU
UGC
UGA
UGG
CGU
CGC
CGA
CGG
AGU
AGC
AGA
AGG
GGU
GGC
GGA
GGG
Cys
Stop
Trp
U
C
A
G
Arg
U
C
A
G
Ser
Arg
Gly*
U
C
A
G
U
C
A
G
T
H
I
R
D
B
A
S
E
©2001 Timothy G. Standish