Chpt 12 Intracellular Compartments anddfd Protein Sorting

8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting

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Intracellular Compartments

and Protein Sorting


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Intracellular Compartments andProtein Sorting

Functionally distinct membrane bound organelles

10 billion proteins of 10,000-20,00 diff kinds

Complex delivery system


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Compartmentalization of Cells

Membranes Partition cell Important cellular functions Impermeable to most hydrophobic molecules contain transport proteins to import and export specific molecules Mechanism for importing and incorporating organelle specific proteins

that define major organelles


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All Eucaryotic Cells Have Same Basic Set of Membrane Bound Organelles


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Major Organelles Nucleus

Cytosol

ER Golgi Apparatus

Mitochondria and Chloroplast

Lysosomes

Endosomes

Peroxisomes


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Occupy 50% cell volume

Perform same basic function

Vary in size and abundance

May take on additional functions

Position dictated by cytoskeleton


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Topology governed by evolutionary originsInvagination of pm creates organelles such as nucleus that aretopologically equivalent to cytosol and communicate via pores


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Topology governed by evolutionary originsEndosymbiosis of mito and plastids creates doublemembrane organelle (have own genome)


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Topology governed by evolutionary originsOrganelles arising from pinching off of pm have interiorequivalent to exterior of cell


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3 Types of Transport Mechanisms

1. Gated Transport:

gated channels

topologically equivalent spaces2. Transmembrane Transport:

protein translocators

topologically distinct space

3. Vesicular transport:membrane enclosed intermediatestopologically equivalent spaces


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2 Types of Sorting Signals in Proteins1. Signal Sequence

continuous sequence of 15-60 aa

sometimes removed from finished protein

sometimes a part of finished protein2. Signal Patch

specific 3d arrangement of atoms on protein surface; aas distant

persist in finished protein


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Signal Sequences/PatchesDirect Proteins to Final Destination

Signal patches direct proteins to:1. nucleus2. lysosomes

Signal Sequences direct proteins to:

1. ER proteins possess N-terminal signal of 5-10 hydrophobic aa2. mito proteins have alternating + chg aa w/ hydrophobic aa3. proxisomal proteins have 3 aa at C-terminus



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Sorting signals recognize complementary sorting receptors Receptors unload cargo Function catalytically and are reusable


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Organelles Cannot be Constructed Denovo

Organelles reproduced via binary fission Organelle cannot be reconstructed from DNA alone Info in form of one protein that pre-exists in organelle mem is required

and passed on from parent to progeny Epigenetic information essential for propogation of cells compartmental

organization


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Transport of Molecules Btwn Nucleus and Cytosol

Nuclear Envelope Two concentric membranes

-Outer membrane contiguous w/ER-Inner membrane contains proteins thatact as

binding sites for chromatin and nuclearlamina

Perforated by nuclear pores for selectiveimport and export


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Nuclear Pore Complex mass of 125 million; ~50 differentproteins arranged in octagon

Typical mammalian cell 3,000-4,000 Contains >1 aqueous channels thruwhich sm molec can readily pass 60,000 cannot pass

Functions ~diaphram Receptor proteins actively transportmolec thru nuclear pore


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Nuclear Localization Signal Generally comprised of two short sequences rich in + chged aa lys & arg Can be located anywhere Thought to form loops or patches on protein surface Resident, not cleaved

Transport thru lg aqueous pores as opposed to translocator proteins Transports proteins in folded state Energy requiring process


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Nuclear Import- the players Importins = cytosolic receptor protein binds to NLS of cargo proteins Nucelar Export Receptors = binds macromolecules to be exported from nucelus Adaptors = sometimes required to bind target protein to nuclear receptor Ran = cytosolic GTP/GDP binding protein complexes with importins in the cytosol. Fibril proteins and nucleoporins contain phenylalanine/glycine repeats (FG) repeats.

Repeats transiently bound and released by importin/cargo/Ran-GDP, causing thecomplex to hop into the nucleus

Import Receptors release cargo in nucleus and return to cytosol

Export Receptors release cargo in cytoplasm and return to nucleus


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Transport of Molecules Btwn Nucleus and CytosolRan GTPase= molecular switch

Drives directional transport in appropriate directin Conversion btwn GTP and GDP bound states mediated by Ran specific regulatory proteinsGAP converts RNA-GTP to Ran-GDP via GTP hydrolysisGEF promotes exchg of GDP for GTP converting Ran-GDP to Ran-GTP

Ran GAP in cytosol thus more Ran-GDP in cytosol Ran GEF in nucleus thus more Ran-GTP in nucleus


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Nuclear Export Works like import in reverse

Export receptors bind export signals and nucleoporins to guidecargo thru pore Import and export receptors member of same gene family


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Regulation Afforded by Access to Transport Machinery Controlling rates of import and export determines steady state location phosphorylation/dephosphorylation of adjacent aa may be required for receptor binding Cytosolic anchor or mask proteins block interaction w/ receptors Protein made and stored in inactive form as ER transmembrane protein


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Control of mRNA Export Proteins w/ export signals loaded onto RNA during transcription and

processing (RNP) Export signals guide RNA out of nucleus thru pores via exportin proteins than

bind RNP

Export mediated by transient binding to FG repeats Imature mRNAs retained by anchoring to transcription and splicing machinery Proteins disassociate in cytosol and return to nucleus


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Nuclear Lamina Meshwork of intermediate filaments

Maintenance of nuclear shape

Spacial organization of nuclear pores

Regulation of transcription

Anchoring of interphase chromatin

DNA replication

Phosphorylation causes depolymerizesduring mitosis when nucleus disassembles


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Nuclear envelop disassembles during mitosis and reassembles

when ER wraps around chromosomes and begins to Fuse


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Protein Transport into theMitochondria and Chloroplast

Subcompartments of the Mitochondria and Chloroplast


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Translocation into Mitochondrial Matrix Governed by: 1. Signal Sequence (amphipathic alpha helix cleaved after import)

2. Protein Translocators

P i T i h


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Players in Protein Translocation of Proteins in Mitochondria TOM- functions across outer membrane TIM- functions across inner membrane OXA- mediates insertion of IM proteins syn w/in mito and helps to

insert proteins initially transported into matrix

Complexes contain components that act as receptors andothers that form translocation channels

P i T i h


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Import of Mitochondrial Proteins

Post-translational Unfolded polypeptide chain

1. precursor proteins bind to receptor proteins of TOM

2. interacting proteins removed and unfolded polypetide is fed into

translocation channel Occurs contact sites joining IM and OM

TOM transports mito targeting signal across OM and once it reaches IMtargeting signal binds to TIM, opening channel complex thru which proteinenters matrix or inserts into IM


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Import of Mitochondrial Proteins Requires energy in form of ATP and H+ gradient and assitance of hsp70

-release of unfolded proteins from hsp70 requires ATP hydrolysis

-once thru TOM and bound to TIM, translocation thru TIM requires

electrochemical gradient

P i T i h


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Protein Transport into IM or IM Space Requires 2 Signal Sequences

1. Second signal =hydrophobic sequence; immediately after 1st signal sequence

2. Cleavage of N-terminal sequence unmasks 2 nd signal used to translocate proteinfrom matrix into or across IM using OXA

3. OXA also used to transport proteins encoded in mito into IM

4. Alternative route bypasses matrix; hydrophobic signal sequence = stoptransfer


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Protein Transport into Chloro Similar to Transport into Mito1. occur posttranslationally2. Use separate translocation complexes in ea membrane3. Translocation occurs at contact sites4. Requires energy and electrochemical gradient5. Use amphilpathic N-terminal signal seq that is removed6. Like the mito a second signal sequence required for translocation

into thylakoid mem or space


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Peroxisomes and Protein Import

Peroxisomes Use O 2 and H 2O2 to carry out oxidative rxns Remove H from specific organic compounds RH 2 + O 2 R + H 2O2 Catalases use H 2O2 to oxidize other substances, particularly in liver and kidney detoxification

H2O2 + RH 2 R + H 2O Beta Oxidation Formation of plasmalogens (abundant class of phospholipids in myelin) Photorespiration and glyoxylate cycle in plants


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Peroxisomes and Protein Import

Peroximsomes in Plants

Site of Photorespiration= glycolate pathway in leaves Called glyoxysomes in seeds where fats converted intosugar


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Proxisomes and Protein Import Peroxisomes arise from pre-existing peroxisomes Signal sequence of 3 aa at COOH end of peroxisomal proteins= import signal Some have signal sequence at N-terminus Involves >23 distinct proteins Driven by ATP hydrolysis Import mechanism distinct, not fully characterized Oligomeric proteins do not unfold when imported Zellweger Disease= peroxisomal deficiency


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ER and Protein Trafficking

Endoplasmic Reticulum Occupies >= 50% of cell volume Continuous with nuclear membrane Central to biosyn macromolecules used to construct other organelles Trafficking of proteins to ER lumen, Gogli, lysosome or those to be secreted

from cell


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ER Central to Protein Synthesis and Trafficking Removes 2 Types of Proteins from Cytosol:

1. transmembrane proteins partly translocated across ER embedded in it

2. water soluble proteins translocated into lumen


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Quantity of SER and ER Dependent Upon Cell TypeRER assoc. w/ protein synthesis

SER assoc. lipid biosynthesis, detoxification, steroid synthesis, Ca2+

storage


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Import of Proteins into ER Occurs co-translationally

Signal recognition sequence recognized by SRP

SRP recognized by SRP receptor

Protein Translocator


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Hydrophobic signal sequence of diff sequence and shape SRP lg hydrophobic pocket lined by Met having unbranched flexible

side chains Binding of SRP causes pause in protein synthesis allowing time for

SRP-ribosome complex to bind to SRP receptor


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Protein to be imported passes through an aqueous pore in

the translocator that is a dynamic structure Sec61 protein translocator

Signal sequence triggers opening of pore

Translocator pore closes when ribo not present


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Some proteins are imported in to ER by a posttranslational mechanism

Proteins released into cytoplasm Binding of chaperone proteins prevents them from folding

Translocation occurs w/out ribo sealing pore

Mechanism whereby protein moves through pore unkwn


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Signal Sequence is Removed from Soluble Proteins Two signaling functions:

1) directs protein to ER membrane2) serves as start transfer signal to open pore

Signal peptidase removes terminal ER signal sequence uponrelease of protein into the lumen


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Folding of ER Resident Proteins ER resident proteins contain an ER

retention signal of 4 specific aa at C-terminus

PDI protein disulfide isomerase oxidizesfree SH grps on cysteines to from disulfidebonds S-S allowing proteins to refold

BiP chaperone proteins, pulls proteinsposttranslationally into ER thru translocator

and assists w/ protein folding


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ER and Protein TraffickingGlycolsylation of ER Proteins Most soluble and transmembrane proteins made in ER are

glycolsylated by addition of an oligosaccharide to Asn Precursor oligosaccharide linked to dolichol lipid in ER mem, in

high energy state

Transfer by oligosaccharyl transferase occurs almost as soon aspolypeptide enters lumen


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Oligosaccharide assembled sugar by sugar onto carrier lipid dolichol


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Retrotranslocation Improperly folded ER proteins are exported and degraded in cytosol Misfolded proteins in ER activate an Unfolded Protein Response to

increase transcription of ER chaperones and degradative enzymes


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The Unfolded Protein Response

ff


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ER and Protein Trafficking Assembly of Lipid Bilayers on ER

ER synthesizes nearly all major classes of lipids Phospholipid synthesis occurs on cytoplasmic face by enzymes in

mem Acyl transferases add two FA to glycerol phosphate producing

phosphatidic acid

Later steps determine head group

ER d P t i T ffi ki g


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Assembly of Lipid Bilayers on ER Scramblase phospholipid translocator equilibrates phospholipids

distribution Flipasses of PM responsible for asymmetric distribution of phospholipids


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Documents

Chpt 12 Intracellular Compartments anddfd Protein Sorting