Deuteronomy 6 5 9 5 And thou shalt

Protein Localization: The Right Part in the Right Place Timothy G. Standish, Ph. D.

DNA Does Not Specify Proteins DNA sequence does not specify protein, but only the

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

Eukaryotic Gene Expression Packaging Cytoplasm Nuclear pores Degradation DNA Transcription Transportation Modification RNA G

After Translation To be effective polypeptide chains must: 1. Fold correctly - This may

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 © 2001

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

Protein Production Mitochondria Leader sequence binding receptor ATP P +ADP Matrix MLSLRQSIRFFKPATRTLCSSRYLL P +ADP

Protein Production Mitochondria Leader sequence binding receptor LS M LR QS IR Peptidease cleaves

Protein Production Mitochondria Leader sequence binding receptor ML SL R Matrix QSI R FFK

Protein Production Mitochondria Leader sequence binding receptor Outer membrane Inner membrane Matrix Inter membrane

Protein Production Mitochondria Leader sequence binding receptor Outer membrane Hsp 60 Matrix Chaperones Inner

Protein Production Mitochondria Leader sequence binding receptor Outer membrane Inner membrane Matrix Mature protein

M S L R Q S I Yeast Cytochrome C Oxidase Subunit IV Leader

Yeast Cytochrome C 1 Leader Charged leader sequence signals for transport to mitochondria First

Protein Production Mitochondria ATP Leader sequence binding receptor P +ADP Outer membrane ATP P

Protein Production Mitochondria Note that chaperones are not involved in folding of proteins in

Leader Sequence Receptors Transporting proteins into the matrix actually involves two receptors, one each

Leader Sequence Receptors Cytosol Intermembrane Matrix T O M 37, 71, 70 TIM 44

Leader Sequence Receptors Cytosol Intermembrane T O M 37, 71, 70 TIM 44 Hsp

Alternative Mechanism � 1. 2. There actually two theories about how the leader operates

Leader Sequence Receptors First part of leader signaling for entrance into mitochondria Cytosol Intermembrane

Leader Sequence Receptors Second part of leader First part of leader signaling for entrance

Leader Sequence Receptors Second part of leader signals for inter membrane space Cytosol Intermembrane

Leader Sequence Receptors Cytosol Intermembrane T O M 37, 71, 70 Second part of

Leader Sequence Receptors Cytosol Intermembrane Matrix Second part of leader signals for inter membrane

Leader Sequence Receptors Cytosol T O M 37, 71, 70 Intermembrane Peptidease Matrix Second

Leader Sequence Receptors Cytosol Intermembrane Peptidease Matrix T O M 37, 71, 70 T

1999 Nobel Prize in Physiology and Medicine �Günter Blobel - For his pioneering work

The Genetic Code Neutral Non-polar Polar Basic Acidic F I U R S C

Slides: 54

Download presentation

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

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

Eukaryotic Gene Expression Packaging Cytoplasm Nuclear pores Degradation DNA Transcription Transportation Modification RNA G AAAAAA Nucleus Export Degradation etc. AAAAAA e m. RNA G som bo Ri RNA Processing 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 Mitochondria Leader sequence binding receptor LS M LR QS IR Peptidease cleaves off the leader Outer membrane FF KP AT RT Inner membrane SS LC RY LL Matrix 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 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 C 1 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 c 1 holds an iron containing heme group and is part of the B-C 1 (III) complex C 1 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 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 Note that chaperones are not involved in folding of proteins in the inter membrane space and that they exist in a low p. H 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 k. D complex ~13. 8 nm across composed of ~9 mostly transmembrane proteins in the outer membrane • Tom 40 Provides the channel for translocation • Tom 5, 6, 7 Are either assembly factors or part of the channel • Tom 20, 22 Recognize most mtproteins via cytosol domains • Tom 37, 70, 71 Receptor for proteins with internal signal sequences 2. TIM - Two complexes on the inner membrane: • Tim 17 -23 Recognizes signal sequence for translocation into the matrix and probably provides the transmembrane channel • Tim 44 Binds both Tim 17 -23 on the matrix side of the inner membrane and Hsp 70 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 T O M 37, 71, 70 TIM 44 Hsp 70 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

Alternative Mechanism � 1. 2. There 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 TIM 44 Hsp 70 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 TIM 44 Hsp 70 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 TIM 44 Hsp 70 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 TIM 44 Hsp 70 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 TIM 44 Hsp 70 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 TIM 44 Hsp 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 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 TIM 44 Hsp 70 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 44 Hsp 70 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 TIM 44 Hsp 70 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

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 UUC UUA UUG CUU CUC CUA CUG Phe 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 T H I R D B A S E © 2001 Timothy G. Standish