Intracellular Compartments and Protein Sorting. ► Functionally distinct membrane bound organelles ► 10 billion proteins of 10,000-20,00 diff kinds ► Complex

Intracellular Intracellular Compartments and Compartments and

Protein SortingProtein Sorting

Intracellular Compartments and Intracellular Compartments and Protein SortingProtein Sorting

►Functionally distinct membrane bound organelles

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

►Complex delivery system

Compartmentalization of Compartmentalization of CellsCells

MembranesMembranes► Partition cell Partition cell ► Important cellular functionsImportant cellular functions► Impermeable to most hydrophobic moleculesImpermeable to most hydrophobic molecules► contain transport proteins to import and export specific contain transport proteins to import and export specific

moleculesmolecules► Mechanism for importing and incorporating organelle specific Mechanism for importing and incorporating organelle specific

proteins that define major organellesproteins that define major organelles

Compartmentalization of CellsCompartmentalization of Cells

All Eucaryotic Cells Have Same Basic Set of Membrane Bound Organelles




Major Organelles

►Nucleus

►Cytosol

►ER

►Golgi Apparatus

►Mitochondria and Chloroplast

►Lysosomes

►Endosomes

►Peroxisomes


►Occupy 50% cell volume

►Perform same basic function

►Vary in size and abundance

►May take on additional functions

►Position dictated by cytoskeleton


Topology governed by evolutionary origins

Invagination of pm creates organelles such as nucleus that are topologically equivalent to cytosol and communicate via pores


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


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


3 Types of Transport Mechanisms

1. Gated Transport:

gated channels

topologically equivalent spaces

2. Transmembrane Transport:

protein translocators

topologically distinct space

3. Vesicular transport:

membrane enclosed intermediates topologically equivalent spaces

Compartmentaliztion of CellsCompartmentaliztion of Cells

Families of Intracellular CompartmentsFamilies of Intracellular Compartments::1.1. nucleus and cytosolnucleus and cytosol2.2. organelles in the secretory pathwayorganelles in the secretory pathway3.3. mitochondriamitochondria4.4. plastidplastid

Transport guided by:Transport guided by:1.1. sorting signals in transported proteins sorting signals in transported proteins

2. complementary receptor proteins2. complementary receptor proteins


2 Types of Sorting Signals in Proteins

1. Signal Sequence

continuous sequence of 15-60 aa

sometimes removed from finished protein

sometimes a part of finished protein

2. Signal Patch

specific 3d arrangement of atoms on protein surface; aa’s distant

persist in finished protein


Signal Sequences/Patches Signal Sequences/Patches

Direct Proteins to Final DestinationDirect Proteins to Final DestinationSignal patches direct proteins to:Signal patches direct proteins to:

1. nucleus1. nucleus

2. lysosomes2. lysosomes

Signal Sequences direct proteins to:Signal Sequences direct proteins to:

1. ER proteins possess N-terminal signal of 5-10 1. ER proteins possess N-terminal signal of 5-10 hydrophobic aahydrophobic aa

2. mito proteins have alternating + chg aa w/ 2. mito proteins have alternating + chg aa w/ hydrophobic aahydrophobic aa

3. proxisomal proteins have 3 aa at C-terminus3. proxisomal proteins have 3 aa at C-terminus



Sorting signals recognize complementary sorting Sorting signals recognize complementary sorting receptorsreceptors

► Receptors unload cargoReceptors unload cargo► Function catalytically and are reusableFunction catalytically and are reusable


Organelles Cannot be Constructed DenovoOrganelles Cannot be Constructed Denovo► Organelles reproduced via binary fission Organelles reproduced via binary fission ► Organelle cannot be reconstructed from DNA aloneOrganelle cannot be reconstructed from DNA alone► Info in form of one protein that pre-exists in organelle mem is Info in form of one protein that pre-exists in organelle mem is

required and passed on from parent to progenyrequired and passed on from parent to progeny► Epigenetic information essential for propogation of cell’s Epigenetic information essential for propogation of cell’s

compartmental organizationcompartmental organization

Transport of Molecules Btwn Nucleus and Transport of Molecules Btwn Nucleus and CytosolCytosol

Nuclear Envelope►Two concentric membranes

-Outer membrane contiguous w/ER -Inner membrane contains proteins that act as binding sites for chromatin and nuclear lamina

►Perforated by nuclear pores for selective

import and export


Nuclear Pore Complex

►mass of 125 million; ~50 different proteins arranged in octagon

►Typical mammalian cell 3,000-4,000

►Contains >1 aqueous channels thru which sm molec can readily pass <5,000; molec > 60,000 cannot pass

►Functions ~diaphram

►Receptor proteins actively transport molec thru nuclear pore



Nuclear Localization SignalNuclear Localization Signal► Generally comprised of two short sequences rich in + chged aa lys & argGenerally comprised of two short sequences rich in + chged aa lys & arg► Can be located anywhereCan be located anywhere► Thought to form loops or patches on protein surfaceThought to form loops or patches on protein surface► Resident, not cleavedResident, not cleaved► Transport thru lg aqueous pores as opposed to translocator proteinsTransport thru lg aqueous pores as opposed to translocator proteins► Transports proteins in folded stateTransports proteins in folded state► Energy requiring processEnergy requiring process

Transport of Molecules Btwn Nucleus and CytosolTransport of Molecules Btwn Nucleus and Cytosol

Nuclear Import- the playersNuclear Import- the players► ImportinsImportins = cytosolic receptor protein binds to NLS of “cargo” proteins = cytosolic receptor protein binds to NLS of “cargo” proteins► Nucelar Export ReceptorsNucelar Export Receptors = binds macromolecules to be exported from = binds macromolecules to be exported from

nucelusnucelus► AdaptorsAdaptors = sometimes required to bind target protein to nuclear receptor = sometimes required to bind target protein to nuclear receptor► RanRan = cytosolic GTP/GDP binding protein complexes with importins in the = cytosolic GTP/GDP binding protein complexes with importins in the

cytosol.cytosol.► Fibril proteins and nucleoporinsFibril proteins and nucleoporins contain phenylalanine/glycine repeats ( contain phenylalanine/glycine repeats (FGFG) )

repeatsrepeats. Repeats transiently bound and released by importin/cargo/Ran-GDP, . Repeats transiently bound and released by importin/cargo/Ran-GDP, causing the complex to “hop” into the nucleuscausing the complex to “hop” into the nucleus

Import Receptors release cargo in nucleus and return to cytosolImport Receptors release cargo in nucleus and return to cytosolExport Receptors release cargo in cytoplasm and return to nucleusExport Receptors release cargo in cytoplasm and return to nucleus


Ran GTPaseRan GTPase= molecular switch = molecular switch ► Drives directional transport in appropriate directinDrives directional transport in appropriate directin► Conversion btwn GTP and GDP bound states mediated by Ran specific regulatory Conversion btwn GTP and GDP bound states mediated by Ran specific regulatory

proteinsproteinsGAP converts RNA-GTP to Ran-GDP via GTP hydrolysisGAP converts RNA-GTP to Ran-GDP via GTP hydrolysisGEF promotes exchg of GDP for GTP converting Ran-GDP to Ran-GTPGEF promotes exchg of GDP for GTP converting Ran-GDP to Ran-GTP

► Ran GAP in cytosol thus more Ran-GDP in cytosolRan GAP in cytosol thus more Ran-GDP in cytosol► Ran GEF in nucleus thus more Ran-GTP in nucleusRan GEF in nucleus thus more Ran-GTP in nucleus


Nuclear ExportNuclear Export► Works like import in reverseWorks like import in reverse► Export receptors bind export signals and nucleoporins to guide Export receptors bind export signals and nucleoporins to guide

cargo thru porecargo thru pore► Import and export receptors member of same gene familyImport and export receptors member of same gene family


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


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

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

bind RNP bind RNP ► Export mediated by transient binding to FG repeatsExport mediated by transient binding to FG repeats► Imature mRNAs retained by anchoring to transcription and splicing machineryImature mRNAs retained by anchoring to transcription and splicing machinery► Proteins disassociate in cytosol and return to nucleusProteins disassociate in cytosol and return to nucleus


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 depolymerizes during mitosis when nucleus disassembles


Nuclear envelop disassembles during mitosis and reassembles when ER wraps around chromosomes and begins to Fuse

Protein Transport into the Protein Transport into the Mitochondria and ChloroplastMitochondria and Chloroplast

Subcompartments of the Mitochondria and Chloroplast


Translocation into Mitochondrial Matrix Governed by:

1. Signal Sequence (amphipathic alpha helix cleaved after import)

2. Protein Translocators


Players in Protein Translocation of Proteins in Players in Protein Translocation of Proteins in MitochondriaMitochondria

► TOM- functions across outer membraneTOM- functions across outer membrane► TIM- functions across inner membraneTIM- functions across inner membrane► OXA- mediates insertion of IM proteins syn w/in mito and helps OXA- mediates insertion of IM proteins syn w/in mito and helps

to insert proteins initially transported into matrixto insert proteins initially transported into matrix

Complexes contain components that act as receptors and Complexes contain components that act as receptors and others that form translocation channelsothers that form translocation channels


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 IM targeting signal binds to TIM, opening channel complex thru which protein enters matrix or inserts into IM


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


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 2nd signal used to translocate protein from 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 = “stop transfer”


Protein Transport into Chloro Similar to Transport into Protein Transport into Chloro Similar to Transport into MitoMito

1.1. occur posttranslationallyoccur posttranslationally2.2. Use separate translocation complexes in ea membraneUse separate translocation complexes in ea membrane3.3. Translocation occurs at contact sitesTranslocation occurs at contact sites4.4. Requires energy and electrochemical gradientRequires energy and electrochemical gradient5.5. Use amphilpathic N-terminal signal seq that is removedUse amphilpathic N-terminal signal seq that is removed6.6. Like the mito a second signal sequence required for Like the mito a second signal sequence required for

translocation into thylakoid mem or spacetranslocation into thylakoid mem or space


Peroxisomes and Protein ImportPeroxisomes and Protein Import

Peroxisomes ►Use O2 and H2O2 to carry out oxidative rxns

►Remove H from specific organic compounds RH2 + O2 R + H2O2

►Catalases use H2O2 to oxidize other substances, particularly in liver and kidney detoxification

H2O2 + R’H2 R’ + H2O

►Beta Oxidation►Formation of plasmalogens (abundant class of phospholipids in myelin)►Photorespiration and glyoxylate cycle in plants

Peroxisomes and Protein ImportPeroxisomes and Protein Import

Peroximsomes in Plants►Site of Photorespiration= glycolate pathway in leaves

►Called glyoxysomes in seeds where fats converted into sugar

Proxisomes and Protein ImportProxisomes 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

ER and Protein TraffickingER and Protein Trafficking

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

secreted from cellsecreted from cell


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


Quantity of SER and ER Dependent Upon Cell Type

RER assoc. w/ protein synthesis

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


Import of Proteins into ER

►Occurs co-translationally

►Signal recognition sequence recognized by SRP

►SRP recognized by SRP receptor

►Protein Translocator


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

flexible side chainsflexible side chains► Binding of SRP causes pause in protein synthesis allowing Binding of SRP causes pause in protein synthesis allowing

time for SRP-ribosome complex to bind to SRP receptortime for SRP-ribosome complex to bind to SRP receptor


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


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

ER and Protein TraffickingER and Protein TraffickingSignal Sequence is Removed from Soluble ProteinsSignal Sequence is Removed from Soluble Proteins► Two signaling functions:Two signaling functions:

1) directs protein to ER membrane1) directs protein to ER membrane

2) serves as “start transfer signal” to open pore2) serves as “start transfer signal” to open pore► Signal peptidase removes terminal ER signal sequence Signal peptidase removes terminal ER signal sequence

upon release of protein into the lumenupon release of protein into the lumen


Single Pass Transmembrane ProteinsSingle Pass Transmembrane Proteins

1.1. N-terminal signal sequence initiates N-terminal signal sequence initiates trans-location and additional hydrophobic trans-location and additional hydrophobic “stop sequence anchors protein in “stop sequence anchors protein in membranemembrane

2.2. Signal sequence is internal and remains Signal sequence is internal and remains in lipid bilayer after release from in lipid bilayer after release from translocatortranslocator

3.3. Internal signal sequence in opposite Internal signal sequence in opposite orientationorientation

4.4. Orientation of start-transfer sequence Orientation of start-transfer sequence governed by distribution of nearby chg governed by distribution of nearby chg aaaa


Multipass Transmembrane ProteinsMultipass Transmembrane Proteins► Combinations of start- and stop-transfer signals determine Combinations of start- and stop-transfer signals determine

topologytopology► Whether hydrophobic signal sequence is a start- or stop-Whether hydrophobic signal sequence is a start- or stop-

transfer sequence depends upon its location in polypeptide transfer sequence depends upon its location in polypeptide chainchain

► All copies of same polypeptide have same orientationAll copies of same polypeptide have same orientation


Folding of ER Resident ProteinsFolding of ER Resident Proteins► ER resident proteins contain an ER ER resident proteins contain an ER

retention signal of 4 specific aa at C-retention signal of 4 specific aa at C-terminusterminus

► PDI protein disulfide isomerase PDI protein disulfide isomerase oxidizes free SH grps on cysteines to oxidizes free SH grps on cysteines to from disulfide bonds S-S allowing from disulfide bonds S-S allowing proteins to refoldproteins to refold

► BiP chaperone proteins, pulls proteins BiP chaperone proteins, pulls proteins posttranslationally into ER thru posttranslationally into ER thru translocator and assists w/ protein translocator and assists w/ protein foldingfolding

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

glycolsylated by addition of an oligosaccharide to Asnglycolsylated by addition of an oligosaccharide to Asn► Precursor oligosaccharide linked to dolichol lipid in ER Precursor oligosaccharide linked to dolichol lipid in ER

mem, in high energy statemem, in high energy state► Transfer by oligosaccharyl transferase occurs almost as Transfer by oligosaccharyl transferase occurs almost as

soon as polypeptide enters lumensoon as polypeptide enters lumen


Oligosaccharide assembled sugar by sugar onto carrier lipid dolichol


RetrotranslocationRetrotranslocation► Improperly folded ER proteins are exported and degraded in cytosolImproperly folded ER proteins are exported and degraded in cytosol► Misfolded proteins in ER activate an “Unfolded Protein Response” Misfolded proteins in ER activate an “Unfolded Protein Response”

to increase transcription of ER chaperones and degradative to increase transcription of ER chaperones and degradative enzymesenzymes


The Unfolded Protein Response

ER and Protein TraffickingER and Protein TraffickingAssembly of Lipid Bilayers on ERAssembly of Lipid Bilayers on ER► ER synthesizes nearly all major classes of lipidsER synthesizes nearly all major classes of lipids► Phospholipid synthesis occurs on cytoplasmic face by Phospholipid synthesis occurs on cytoplasmic face by

enzymes in memenzymes in mem► Acyl transferases add two FA to glycerol phosphate Acyl transferases add two FA to glycerol phosphate

producing phosphatidic acid producing phosphatidic acid ► Later steps determine head groupLater steps determine head group


Assembly of Lipid Bilayers on ERAssembly of Lipid Bilayers on ER►Scramblase phospholipid translocator equilibrates Scramblase phospholipid translocator equilibrates phospholipids phospholipids distributiondistribution►Flipasses of PM responsible for asymmetric distribution of Flipasses of PM responsible for asymmetric distribution of phospholipidsphospholipids


Phospholipid Exchange Proteins

►Transfer individual phosphlipids between membranes at random btwn all membranes

►Exchange protein specificity

►Extracts phospholipid and diffuses away w/ it buried w/in lipid binding site; discharges phospholipid when it encounters another membrane


Documents

Intracellular Compartments and Protein Sorting. ► Functionally distinct membrane bound organelles ► 10 billion proteins of 10,000-20,00 diff kinds ► Complex