Deuteronomy 6:5-9

©2001 Timothy G. Standish

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.


Protein Protein Localization:Localization:

The Right Part in the Right PlaceThe Right Part in the Right Place

Timothy G. Standish, Ph. D.


DNA Does Not Specify ProteinsDNA Does Not Specify ProteinsDNA 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.


3’

5’

5’

3’

Transcription And Translation Transcription And Translation In ProkaryotesIn Prokaryotes

Ribosome

Ribosome5’

mRNA

RNAPol.


Cytoplasm

G AAAAAA

Export

Degradation etc.G AAAAAA

Eukaryotic Gene ExpressionEukaryotic Gene Expression

G AAAAAA

RNAProcessing

mRNA

RNA

Transcription

DNA

Nucleus

Nuclear pores

Ribosom

e

Translation

Packaging

Modification

Transportation

Degradation


After TranslationAfter 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 amion acids

3. Be in the correct location - Which can be, as we shall see, a complex process


Cytoplasm

Nucleus

Protein Production and Protein Production and TransportTransport

Endoplasmic Reticulum

Ribosomes

Smooth

Rough

Gogi Complex


Cytoplasm

Nucleus

Protein Production Protein Production Mitochondria and ChloroplastsMitochondria and Chloroplasts

G AAAAAA

Export

ChloroplastMitochondrion


Cytoplasm

Nucleus

ChloroplastMitochondrion

Protein Production Protein Production Mitochondria and ChloroplastsMitochondria and Chloroplasts


Protein Production Protein Production MitochondriaMitochondria

Matrix

Inter membrane space

Inner membraneOuter membrane



Matrix

Inter membrane

space

Inner membrane

Outer membrane

Leader sequence binding receptor

ML

SLR

QS

IRF

FK

PA

TR

TL

CS

SRY

LL

P +ADP

ATP

P +ADP

ATP

©2001 Timothy G. StandishMatrix

Inner membrane

Outer membrane

MLSLR

QSIR

FFKPA

TRTLC

SSRY

LL

Inter membrane

space



Peptidease cleaves off the leader



Matrix

Inner membrane

Outer membrane

MLSLRQSIRFFKPATRTLCSSRYLL

Inter membrane

space




Matrix

Inner membrane

Outer membrane

Inter membrane

space




Matrix

Inner membrane

Outer membrane

Inter membrane

space


Hsp60

Hsp60

Chaperones



Matrix

Inner membrane

Outer membrane

Inter membrane

space


Mature protein


Yeast Cytochrome C Yeast Cytochrome C Oxidase Subunit IV LeaderOxidase Subunit IV Leader

MLSLRQSIRFFKPATRTLCSSRYLL

RY

PL

T

CS

R

L

S

T

I

KP

R

F

A

F

M

RQ

L

L

S

S

This leader sequence probably forms an 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

First 12 residues are sufficient for transport to the mitochondria

Neutral Non-polarPolarBasicAcidic


Yeast Cytochrome C1 LeaderYeast Cytochrome C1 Leader

MFSNLSKRWAQRTLSKTLKGSKSAAGTATSYFE-KLVTAGVAAAGITASTLLYANSLTAGA--------------

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


Second cut

First cut

Uncharged second leader sequence signals for transport accross inner membrane into the intermembrane space

Charged leader sequence signals for transport to mitochondria



Matrix

Inter membrane space

Inner membraneOuter membrane



Matrix

Inter membrane

space

Inner membrane

Outer membrane


P +ADP

ATP

P +ADP

ATP

Peptidease cleaves off the leader



Matrix

Inter membrane

space

Inner membrane

Outer membrane




Matrix

Inter membrane

space

Inner membrane

Outer membrane


Mature protein

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 ReceptorsLeader Sequence ReceptorsTransporting 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• Tom37,70,71Receptor 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.


Matrix

Intermembrane

Cytosol

Inner membraneInner membrane

Outer membraneOuter membrane

MGE

TI

M17-23

TI

M17-23

TOM

5,6,7

TOM

5,6,7

TOM40

TOM40

TIM44TIM44

Hsp70

TOM22,20

TOM22,20

TOM37,

71,70

TOM37,

71,70

Leader Sequence ReceptorsLeader Sequence Receptors


MGE


TI

M17-23

TI

M17-23

TIM44TIM44

Hsp70Matrix

Cytosol

Intermembrane


Outer membraneOuter membraneTOM

5,6,7

TOM

5,6,7

TOM22,20

TOM22,20

TOM40

TOM40

TOM37,

71,70

TOM37,

71,70


MGE

TI

M17-23

TI

M17-23

TIM44TIM44

Hsp70Matrix

Cytosol

Intermembrane



5,6,7

TOM

5,6,7

TOM22,20

TOM22,20

TOM40

TOM40

TOM37,

71,70

TOM37,

71,70



MGE

TI

M17-23

TI

M17-23

TIM44TIM44

Hsp70

Matrix

Cytosol

Intermembrane



5,6,7

TOM

5,6,7

TOM22,20

TOM22,20

TOM40

TOM40

TOM37,

71,70

TOM37,

71,70



MGE

TI

M17-23

TI

M17-23

TIM44TIM44

Hsp70

Matrix

Cytosol

Intermembrane



5,6,7

TOM

5,6,7

TOM22,20

TOM22,20

TOM40

TOM40

TOM37,

71,70

TOM37,

71,70


Peptidease


MGE

TI

M17-23

TI

M17-23

TIM44TIM44

Hsp70

Matrix

Cytosol

Intermembrane



5,6,7

TOM

5,6,7

TOM22,20

TOM22,20

TOM40

TOM40

TOM37,

71,70

TOM37,

71,70



MGE

TI

M17-23

TI

M17-23

TIM44TIM44

Hsp70

Matrix

Cytosol

Intermembrane



5,6,7

TOM

5,6,7

TOM22,20

TOM22,20

TOM40

TOM40

TOM37,

71,70

TOM37,

71,70


Hsp60


MGE

TI

M17-23

TI

M17-23

TIM44TIM44

Hsp70

Matrix

Cytosol

Intermembrane



5,6,7

TOM

5,6,7

TOM22,20

TOM22,20

TOM40

TOM40

TOM37,

71,70

TOM37,

71,70



Alternative MechanismAlternative 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.


MGE


TI

M17-23

TI

M17-23

TIM44TIM44

Hsp70Matrix

Cytosol

Intermembrane



5,6,7

TOM

5,6,7

TOM22,20

TOM22,20

TOM40

TOM40

TOM37,

71,70

TOM37,

71,70

First part of leader signaling for entrance into mitochondria


MGE

TI

M17-23

TI

M17-23

TIM44TIM44

Hsp70Matrix

Cytosol

Intermembrane



5,6,7

TOM

5,6,7

TOM22,20

TOM22,20

TOM40

TOM40

TOM37,

71,70

TOM37,

71,70


Second part of leader signals for inter membrane space



MGE

TI

M17-23

TI

M17-23

TIM44TIM44

Hsp70

Matrix

Cytosol

Intermembrane



5,6,7

TOM

5,6,7

TOM22,20

TOM22,20

TOM40

TOM40

TOM37,

71,70

TOM37,

71,70

Leader Sequence ReceptorsLeader Sequence ReceptorsFirst part of leader signaling for entrance into mitochondria



MGE

TI

M17-23

TI

M17-23

TIM44TIM44

Hsp70

Matrix

Cytosol

Intermembrane



5,6,7

TOM

5,6,7

TOM22,20

TOM22,20

TOM40

TOM40

TOM37,

71,70

TOM37,

71,70 First part of leader signaling for entrance into mitochondria




MGE

TI

M17-23

TI

M17-23

TIM44TIM44

Hsp70

Matrix

Cytosol

Intermembrane



5,6,7

TOM

5,6,7

TOM22,20

TOM22,20

TOM40

TOM40

TOM37,

71,70

TOM37,

71,70





MGE

TI

M17-23

TI

M17-23

TIM44TIM44

Hsp70Matrix

Cytosol

Intermembrane



5,6,7

TOM

5,6,7

TOM22,20

TOM22,20

TOM40

TOM40

TOM37,

71,70

TOM37,

71,70



Second part of leader prevents entrance into TIM 17-23



MGE

TI

M17-23

TI

M17-23

TIM44TIM44

Hsp70Matrix

Cytosol

Intermembrane



5,6,7

TOM

5,6,7

TOM22,20

TOM22,20

TOM40

TOM40

TOM37,

71,70

TOM37,

71,70



First part of leader signaling for entrance into

mitochondria


Peptidease


MGE

TI

M17-23

TI

M17-23

TIM44TIM44

Hsp70Matrix

Cytosol

Intermembrane



5,6,7

TOM

5,6,7

TOM22,20

TOM22,20

TOM40

TOM40

TOM37,

71,70

TOM37,

71,70



Peptidease


Peptidease

Polypeptide passes through TOM, but not TIM


MGE

TI

M17-23

TI

M17-23

TIM44TIM44

Hsp70Matrix

Cytosol

Intermembrane



5,6,7

TOM

5,6,7

TOM22,20

TOM22,20

TOM40

TOM40

TOM37,

71,70

TOM37,

71,70


Peptidease

Protein localized in the intermembrane space


1999 Nobel Prize in Physiololgy 1999 Nobel Prize in Physiololgy and Medicineand Medicine

Günter Blobel - For his pioneering work in discovery of signal sequences, the molecular zip codes of protein production and localization


S E C O N D B A S E

A

GGUGGCGGAGGG

Gly*

AGUAGCAGAAGG

Arg

G

CGUCGCCGACGG

Arg

GUGUUGCUGAUGG

C

GAUGACGAAGAG

AAUAACAAAAAG

Glu

CAUCACCAACAG

AUAUUACUAAUAG

Stop

Tyr

GUUGUCGUAGUG

Val

AUUAUCAUAAUG start

Ile

CUUCUCCUACUG

Leu

UUUUUUCUUAUUG

Leu

Phe

Met/

GCUGCCGCAGCG

Ala

ACUACCACAACG

Thr

CCUCCCCCACCG

Pro

CUCUUCCUCAUCG

Ser

UCAG

U

UCAG

UCAG

UCAG

Gln†

His

Trp

Cys THIRD

BASE

FIRST

BASE

The Genetic CodeThe Genetic Code

Asp

Lys

Asn†

Stop

Ser


†Have aminegroups

*Listed as non-polar bysome texts

Deuteronomy 6:5-9

Documents

Transcript of Deuteronomy 6:5-9