Powerpoint 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.

Transcription And Translation
In Prokaryotes
5’ 3’
3’ 5’
RNA
Pol.
Ribosome
mRNA
Ribosome
5’

Eukaryotic Gene Expression
Nuclear
Cytoplasm
Packaging
pores Degradation
DNA
Transcription
RNA
Transportation
Modification
RNA
Processing
Ri
Degradation etc.
bo
s om
mRNA G AAAAAA G AAAAAA
e
Export Translation
Nucleus

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
Protein Production and
Transport
Ribosomes
Cytoplasm
Rough
Nucleus
Endoplasmic
Reticulum
Smooth
Gogi
Complex

Protein Production
Mitochondria and Chloroplasts
Cytoplasm
Nucleus G AAAAAA
Export
Mitochondrion Chloroplast

Protein Production
Mitochondria and Chloroplasts
Cytoplasm
Nucleus
Mitochondrion Chloroplast

Protein Production
Mitochondria
Outer membrane
Inner membrane
Matrix
Inter membrane space

Protein Production
Mitochondria
Leader sequence
ATP binding receptor
Outer membrane
P +ADP
ATP MLSLRQSIRFFKPATRTLCSSRYLL
P +ADP
Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
M
LS
Peptidease LR
cleaves off QS
the leader
RFI
FK
PA
Inner membrane
TR
TL
Inter
CS
membrane
S RY
Matrix space
LL

Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
ML
SL RQ
SIR
FFK Inner membrane
PA
T RT Inter
LC
SSR membrane
YL
Matrix L space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Hsp60
Hsp60
Inner membrane
Inter
Chaperones membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Inter
membrane
Matrix Mature protein
space
M
L Yeast Cytochrome C
S
L
R
Oxidase Subunit IV Leader
Q Neutral Non-polar
S First 12 residues are sufficient for Polar
I Basic
R transport to the mitochondria
Acidic
F
F MLSLRQSIRFFKPATRTLCSSRYLL
K
P
T
A  This leader sequence probably forms an
R
 helix
T
L  This would localize specific classes of
C
S amino acids in specific parts of the helix
S
R  There are about 3.6 amino acids per turn
Y
L of the helix with a rise of 0.54 nm per
P turn ©2001 Timothy G. Standish
Yeast Cytochrome C1 Leader
Charged leader sequence signals
for transport to mitochondria First cut
MFSNLSKRWAQRTLSKTLKGSKSAAGTATSYFE-
KLVTAGVAAAGITASTLLYANSLTAGA--------------
Second cut
Uncharged second leader sequence signals for transport
accross inner membrane into the intermembrane space Neutral Non-polar
 Cytochrome c functions in electron transport and is thus Polar
Basic
associated with the inner membrane on the Acidic
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
Protein Production
Mitochondria
Outer membrane
Inner membrane
Matrix
Inter membrane space

Protein Production
Mitochondria
Leader sequence
ATP binding receptor
Outer membrane
P +ADP
ATP
P +ADP
Peptidease
cleaves off
the leader Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Note that chaperones are not
involved in folding of proteins Leader sequence
in the inter membrane space binding receptor
and that they exist in a low pH Outer membrane
environment
Inner membrane
Mature protein
Inter
membrane
Matrix space
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
Cytosol
T T T T
O O O O Outer membrane
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane
T
I Inner membrane
M
17-
23
TIM44
Matrix Hsp70
MGE
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane
T
I Inner membrane
M
17-
23
TIM44
Matrix Hsp70
MGE
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane
T
I Inner membrane
M
17-
23
TIM44
Matrix Hsp70
MGE
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane T
I
M
17-
23
TIM44 Inner membrane
Hsp70
MGE
Matrix
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane T
I
M
17-
23
Hsp70
MGE
Matrix
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane T
I
M
17-
23
Hsp70
MGE
Matrix
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane T
I
M
17-
23
Hsp70
MGE
Peptidease
Matrix
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane T
I
M
17-
23
Hsp70
MGE
Pep
Matrix ti d ea
se
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane T
I
M
17-
23
Hsp70
MGE
Matrix
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane T
I
M
17-
23
Hsp70
MGE
Matrix
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane T
I
M
17-
23
Hsp70
MGE
Matrix
Hsp60
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane T
I
M
17-
23
Hsp70
MGE
Matrix
Alternative Mechanism
 There are actually two theories about how the
leader operates to localize mtproteins in the inter
membrane space:
1. The first, as shown in the previous slides,
involves the whole protein moving into and then
out of the matrix
2. 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.
First part of leader signaling for
entrance into mitochondria
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane
T
I Inner membrane
M
17-
23
TIM44
Matrix Hsp70
MGE
Leader
Second part of leader
Sequence Receptors
signals for inter membrane space entrance into mitochondria
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane
T
I Inner membrane
M
17-
23
TIM44
Matrix Hsp70
MGE
Leader
Second part of leader
Sequence Receptors
signals for inter membrane space entrance into mitochondria
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane T
I
M
17-
23
Hsp70
MGE
Matrix
Leader Sequence
Second part of leader signals
Receptors
for inter membrane space
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
First part of leader signaling for entrance
Intermembrane T into mitochondria
I
M
17-
23
Hsp70
MGE
Matrix
Leader Sequence
Second part of leader signals
Receptors
for inter membrane space
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane T
I
M
17-
23
Hsp70 First part of
MGE
leader signaling for
entrance into mitochondria
Matrix
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane
Second part of leader signals Second part of leader prevents
for inter membrane space entrance into TIM 17-23
T
I Inner membrane
M
17-
23
TIM44
Matrix Hsp70
MGE entrance into mitochondria
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane
Second part of leader prevents
entrance into TIM 17-23
T
I Inner membrane
M
17-
23 First part of leader signaling
TIM44
Matrix Hsp70
for entrance into
Second part of leader signals MGE
mitochondria
for inter membrane space Peptidease ©2001 Timothy G. Standish
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane Polypeptide
Second part of leader passes
prevents
through TOM, but
Peptidease entrance into TIM 17-23
not TIM
T
I Inner membrane
M
17-
23
TIM44
Matrix Hsp70
Second part of leader signals MGE
for inter membrane space Peptidease ©2001 Timothy G. Standish
Cytosol
T T T T
M M M M
37, 40 5,6,722,
71,70 20
Intermembrane
Protein localized in the
Peptidease intermembrane space
T
I Inner membrane
M
17-
23
TIM44
Matrix Hsp70
MGE
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

The Genetic Code
Neutral Non-polar
Polar SECOND BASE
Basic
Acidic U C A G
F UUU
UUC
Phe
UCU
UCC
UAU
UAC
Tyr
UGU
UGC
Cys U
C
T
I U UUA Leu UCA
Ser
UAA Stop UGA Stop A H
UUG UCG UAG UGG Trp G
R CUU CCU CAU CGU U
I
His
S C CUC
CUA
Leu CCC
CCA
Pro CAC
CAA
CGC
CGA
Arg C
A
R
Gln †
T CUG CCG CAG CGG G D
AUU ACU AAU AGU U
AUC Ile ACC AAC
Asn† AGC
Ser C
†Have amine B A AUA ACA
Thr
AAA Lys AGA Arg A B
AUGMet/start ACG AAG AGG G
groups
A GUU GCU GAU GGU U
A
Asp
*Listed as
S G GUC
GUA
Val GCC
GCA
Ala GAC
GAA
GGC
GGA
Gly* C
A
S
Glu
non-polar by E GUG GCG GAG GGG G E
some texts

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Deuteronomy 6:5-9

5 And thou shalt love the LORD thy God with

©2001 Timothy G. Standish

©2001 Timothy G. Standish

©2001 Timothy G. Standish

©2001 Timothy G. Standish

©2001 Timothy G. Standish

©2001 Timothy G. Standish

©2001 Timothy G. Standish

Inter membrane space

©2001 Timothy G. Standish

©2001 Timothy G. Standish

Inter membrane space

©2001 Timothy G. Standish

©2001 Timothy G. Standish

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