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Protein Targeting.doc

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University of Ottawa
Kathleen Gilmour

Protein Targeting Why is targeting important? The Lifecycle of a Protein -protein synthesis occurs on ribosomes -when proteins are released from the ribosome, their fate is till uncertain -we can organize what happens to them into 3 conceptual areas: 1) They will need to get to the right place in order to function 2) They will undergo modification (>80% are covalently modified) 3) They will be removed from the cell (i.e.: if there are mistakes) -protein trafficking / sorting / targeting is important because proteins must go to the right place in order to function -proteins do not function without context -they interact with other macromolecules and within the correct context (within the correct part of the cell) -proteins achieve context by participating in reactions (i.e.: metabolic functions; proteins involved in photosynthesis must go into the chloroplast to interact with all the other proteins involved with photosynthesis) -signals / tags direct the proteins -signals are part of the amino acid sequence that are recognized by a receptor -the model always involves a signal and a receptor -all proteins (except proteins made in organelles) are translated in the cytoplasm Examples of Protein Targeting and Mechanisms 1) endoplasmic reticulum -rough ER = granulation = more protein export -rough ER is involved in the export of proteins -rough ER is a consequence of the simultaneous synthesis and transportation of proteins 2) Apoplast -describes the outside of the cell in plants (between the cell wall and the membrane) 3) mitochondrion 4) chloroplast 5) nucleus General Principles for Protein Targeting -in E.coli, the newly synthesized protein must get to its proper location but in eukaryote, targeting is more complicated -there are fewer locations / final destinations for the protein to go in E.coli -in eukaryotes, there are many more destinations for the protein to go -the eukaryote has internal endomembrane system organelles and nucleus (e.g.: ER, golgi structures, and other vesicles) -in eukaryotes the organelles of the endomembrane system include: the nuclear envelope, the endoplasmic reticulum, the Golgi apparatus, lysosomes, vacuoles, vesicles, peroxisomes and the cell membrane -the internal structure is complex and there is constant movement within the cell -the solution is to use information in the protein as a signal for transport across the membrane using specific receptors -since proteins fold up, signals are at the end of a protein -the amino terminal end (most commonly) or the carboxyl terminal end are usually not folded up -the steps involved in the lifecycle of the protein: 1) Translation 2) Interaction with the receptor and unfolding -signals on protein allow it to bind to the receptor (i.e.: a signal peptide) -signal is typically on the amino-terminal end -in order to go through the receptor, the protein must unfold -proteins aid unfolding (i.e.: chaperones) 3) Translocation 3) Refolding / Processing -proteases may remove signals (i.e.: signal peptidases, signal proteases, etc.) -proteins aid unfolding (i.e.: chaperones) -these steps get the protein through one membrane -some proteins get inside the nucleus without a signal by binding onto another protein with a signal Protein targeting in the Endoplasmic Reticulum (ER) -the ER is the most commonly studied example of protein transport -other examples may include mitochondrion, outside the cell (i.e.: the apoplast in plants), the chloroplast, the nucleus, golgi structures, and other vesicles -involves co-translation transport -transport is occurring as translation goes on -transport across the membrane in the ER (the lumen) is the first step for targeting to many locations (i.e.: may stay in the ER, go to golgi, vesicles, and the outside of the cell via the vesicles) -a signal peptide and a receptor is required -leads to the partitioning into many different organelles after it enters the ER through modifications -in the absence of further information in the amino acid sequence , after going into the ER, the protein is exported to the outside of the cell -this is referred to as the default pathway -exported via vesicles and the golgi Signal Mechanism in the ER: Co-translational Transport (pg 315. figure 14-19) -the message is being transported as it is translated (i.e.: co-translational) -message protein starts off with a poly-A tail and 5'-cap -when process begins, the amino terminal is made first and the signal is eventually exposed -this makes sense because if it was carboxy-terminal, you would have to finish protein before you can translocate it so it wouldn’t be co-translational 1) Signal peptide emerges from the ribosome -signal recognition particle (SRP) binds and translation stops / pauses 2) SRP binds to the SRP receptor (the docking protein) -translation resumes -the polypeptide enters the rough ER lumen and binds to the signal peptidase 3) Signal peptidase cleaves the signal from the growing polypeptide -the mature amino-terminal of the protein is exposed -the rest of the protein is getting translated 4) Translation of the mRNA is complete and is put into the ER -ribosomal subunits are about to dissociate -the signal peptide for ER transport is a length of 20-50 amino acids with a hydrophobic core -these are recognized by the signal recognition particle and the complex binds to the SRP receptor -when translation resumes, the signal peptide is cleaved by the signal peptidase -the original protein is referred to as the preprotein -a protein follows, then a pre-proprotein and a proprotein, then a mature protein -e.g.: prelysozyme becomes lysozyme when the signal peptide is removed -inside the ER, various events occur: -the majority of the proteins are modified in the same way; they must then, either go through the endomembrane system or stay there -if the proteins' fate is to stay in the endomembrane system, they have an extra signal called the retention signal -in the lumen of the ER: -proper folding occurs with the aid of chaperones -if proteins do not fold properly, a retrograde signal is sent to stop the transcription of the proteins -addition of complex sugars to the motif occurs -this is known as N-linked glycosylation since the side chain of R is the site of modification -sugars are a
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