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Shore 29-34.doc

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McGill University
BIOC 312
Joel Shore

Lecture 29 Protein Topogenesis Less emphasis on the memorization of the material, more emphasis on the underlying principles and strategies. The functionality and complexity of protein translation products can be increased beyond the encoded 20,000 proteins of the cell through protein post-translational modification o 60-70 different chemical functionalities and processing can be directed towards proteins Protein Topogenesis Topo: Topology; Genesis: To make Topogenesis refers to the selective partitioning of a particular protein to its final destination Proteins in eukaryotes are directed towards different locations and strctures within the cell: o mRNA from the nucleus is translated on cytoplasmic ribosomes and then there is differentiation and compartmentalization of proteins based on function o Eukaryotes are characterized by the amount of differentiation at the level of structure and function Differs from prokaryotes which are considered to be bags of proteins and DNA Properties of eukaryotes: o Chromatin is segregated from the protein synthetic machinery o Chromatin has two different strcutres: Euchromatin (white) which is expressed Heterochromatin (dark) which is silent o RNA is exported out of the nucleus through pores and undergoes translation o Matabolic pathways are compartmentalized allowing for execution of different functions: Ex. respiration in mitochondria, oxidation in peroxisomes, protein segregation in the Endoplasmic Reticulum (ER) How are proteins synthesized in the common site of the cytoplasm partitioned to specific different organelles and structures within the cell and integrated with proper orientation? o There are a number of simple underlying principles that have driven the evolution of this pathway/process Mechanisms of segregation is driven by two predominant important factors: o Specificity: Proteins that go to the ER only go to the ER, they do not usually go to the nucleus or mitochondria Some proteins are partitioned between two organelles, but there is a different underlying mechanism for this This relates to common proteins that are found in several organelles o Thermodynamics: Deals with the problem of partitioning an extremely hydrophilic protein between/through a hydrophobic lipid bilayer Protein must translocate this membrane in order to become part of the organelle or insert into the bilayer Evolution has had to deal with specificity and thermodynamics. Mechanisms have evolved to take these into account 5 organelles have evolved mechanisms to take proteins from the cytoplasm:ER, Nucleus, Mitochondria, Chloroplasts (plants) and peroxisomes BIOC 312 84 o Cell membrane and Golgi are not included as organelles that are competent for selective uptake of proteins because they get their proteins through another organelle Topogenic Sequences direct proteins to their final destination o Topogeneic sequences are encoded in the primary sequence of the protein o Signal sequence ( or targeting sequence) direct the protein to the right organelle Additional sequences and folding structures get the protein to its final destination: o These sequences and regions include: Memebrane anchor, Stop transfer sequences and sorting sequences Stop transfer sequences are used for proteins that go into membranes and selects membrane orientation The ER is the starting point of the secretory pathway: o Secretory pathway goes from: ER Golgi vesicles targeted for membrane fusion of vesicles with the plasma membrane o The continuum of space goes from the interior of the ER to the outside of the cell o Vesicles can bifurcate: Some can go to lysosomes Some can go to other structures within the cell The secondary sequence zip code determines its final location within the cell The study in this field began in the late 1970s: o Eukaryotic cells were burst open and homogenized Large sub-cellular structures like the mitochondria would remain intact o The ER breaks down from continuous sheets seen in the cell to form small vesicles Note: The ER contains a subpopulation of ribosomes that are associated with the membrane, membrane bound ribosomes o These vesicles can be isolated with the ribosomes and assocated RNA removed o Vesciles were combined with different translational products and different chemical approaches were used to conduct in vitro reconstitution experiments to deduce the underlying mechanism o As technology and understanding increases you can: Express individual proteins Introduce mutations into the proteins Introduce specific functionality within the amino acids (ex. Fluorescent probes) Moves away from vesicles to synthetic liposomes Work with high purity products Impose high level structure determination of the components at the atomic level o We now know a lot about what is going on, but there is still a lot to know Information was gained through biochemical and genetic studies: o In vitro reconstitution by classical biochemical procedures was used: Rate of progression in elucidating these pathways is directly proportional to the quality of assays available The better the assays, the better the elucidations o Genetics showed that protein topogenesis is a highly conserved process Mechanisms are largely conserved from yeast to man Most information has been achieved through the study of yeast, this information can be translated to higher eukaryotes Signal Sequences Signal Sequences are associated with proteins that are destined for the five organelles that are competent for protein uptake BIOC 312 85 Signal sequences are not sequence specific, they have general characteristics o There are different signals allowing for different specificity ER: o Comprises mainly of the amino (NH ) t2rminus o A stretch of 30 amino acids that has a hydrophobic core of 6-12 amino acids o Typically preceded by basic amino acids (positively charged ) ex. Lysine or Arginine Mitochondria: o Stretch of basic amino acids o Lacks the hydrophobic core Chloroplasts: o Hard to come up with characteristics o Contain some hydrophobic amino acids poor in negatively charged amino acids Peroxisomes: o A lot of the peroxisome proteins have a specific tri-peptide sequence: Serine-Lysine-Leusine (SKL) located at the extreme C-terminus o Not present in all peroxisome proteins Nucleus o Contains Nuclear localization sequence (NLS) o Bifercated cluster of positively charged amino acids The individual sequences are less important than the folding of these regions into common structures o Signal sequences for each organelle fold into a common and unique structure o With these types of characteristics you could presumably get 300 proteins that have to go to the mitochondria to adopt a common conformation which then imposes specificity for targeting Steps in Protein Topogenesis: These principles extend for protein degradation, apoptosis pathways and signal transduction pathways #1. Targeting information residues in topogenic sequences. o Signal sequences are often located towards the N-terminus of the protein Encodes information for targeting Peroxisome signal sequences are an exception, they are located at the C-terminus o Targeting information relies on these topogenic sequences o Membrane anchor, stop-transfer sequences and sorting sequences are distributed throughout the primary sequence of the amino acid Tell the protein to adopt a certain structure and provides specificity o Determination of a signal sequences: Use recombinant structures where the signal sequence has been removed and reintroduce truncated protein back into a cell Look to see if it goes to the mitochondria If the proteins no longer proceeds to the mitochondria, then this implies that the sequence is important for targeting BIOC 312 86 Corollary experiment: Place the signal sequence on a known cytosolic protein and observe if the protein is re-targeted to the mitochondria Re-taregtting is generally observed These two experiments show that signal sequences are necessary and sufficient. Signal sequences are the only entities required The facts that you could put this sequence on a cytosolic protein indicates that the process is fairly passive The rest of the process with respect to the protein is fairly passivein nature Protein structures are competent to be taken by targeting sequences to the final destination Further corollary: Ask the question, what is the default pathway of a protein targeted towards a particular organelle? Ex. The mitochondria has four compartments. What is the pathway that a signal sequence is going to take a protein independent of any other information in the protein. Fuse the target sequen
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