Intracellular Compartments and Protein Sorting

► Functionally distinct membrane bound organelles ► 10 billion proteins of 10,000-20,00 diff kinds ► Complex delivery system

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

Compartmentalization of Cells All Eucaryotic Cells Have Same Basic Set of Membrane Bound Organelles

Compartmentalization of Cells

Major Organelles ► Nucleus ► Cytosol ► ER ► Golgi Apparatus ► Mitochondria and Chloroplast ► Lysosomes ► Endosomes ► Peroxisomes

Compartmentalization of Cells ► Occupy 50% cell volume ► Perform same basic function ► Vary in size and abundance ► May take on additional functions ► Position dictated by cytoskeleton

Compartmentalization of Cells Topology governed by evolutionary origins Invagination of pm creates organelles such as nucleus that are topologically equivalent to cytosol and communicate via pores

Compartmentalization of Cells Topology governed by evolutionary origins Endosymbiosis of mito and plastids creates double membrane organelle (have own genome)

Compartmentalization of Cells Topology governed by evolutionary origins Organelles arising from pinching off of pm have interior equivalent to exterior of cell

Compartmentalization of Cells 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 Cells Families of Intracellular Compartments: 1.nucleus and cytosol 2.organelles in the secretory pathway 3.mitochondria 4.plastid Transport guided by: 1. sorting signals in transported proteins 2. complementary receptor proteins 2. complementary receptor proteins

Compartmentalization of Cells 2 Types of Sorting Signals in Proteins 1. Signal Sequence continuous sequence of 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

Compartmentalization of Cells

Signal Sequences/Patches Direct Proteins to Final Destination Signal patches direct proteins to: 1. nucleus 2. lysosomes Signal Sequences direct proteins to: 1. ER proteins possess N-terminal signal of 5-10 hydrophobic aa 2. mito proteins have alternating + chg aa w/ hydrophobic aa 3. proxisomal proteins have 3 aa at C-terminus Compartmentalization of Cells

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

Compartmentalization of Cells 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 cell’s compartmental organization

Transport of Molecules Btwn Nucleus and Cytosol 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

Transport of Molecules Btwn Nucleus and Cytosol 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 60,000 cannot pass ► Functions ~diaphram ► Receptor proteins actively transport molec thru nuclear pore

Transport of Molecules Btwn Nucleus and Cytosol

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

Transport of Molecules Btwn Nucleus and Cytosol 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 the complex 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

Transport of Molecules Btwn Nucleus and Cytosol Ran GTPase= molecular switch ► Drives directional transport in appropriate directin ► Conversion btwn GTP and GDP bound states mediated by Ran specific regulatory proteins GAP converts RNA-GTP to Ran-GDP via GTP hydrolysis GEF 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

Transport of Molecules Btwn Nucleus and Cytosol Nuclear Export ► Works like import in reverse ► Export receptors bind export signals and nucleoporins to guide cargo thru pore ► Import and export receptors member of same gene family

Transport of Molecules Btwn Nucleus and Cytosol 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

Transport of Molecules Btwn Nucleus and Cytosol 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

Transport of Molecules Btwn Nucleus and Cytosol 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

Transport of Molecules Btwn Nucleus and Cytosol Nuclear envelop disassembles during mitosis and reassembles when ER wraps around chromosomes and begins to Fuse

Protein Transport into the Mitochondria and Chloroplast Subcompartments of the Mitochondria and Chloroplast

Protein Transport into the Mitochondria and Chloroplast Translocation into Mitochondrial Matrix Governed by: 1. Signal Sequence (amphipathic alpha helix cleaved after import) 2. Protein Translocators

Protein Transport into the Mitochondria and Chloroplast 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 and others that form translocation channels

Protein Transport into the Mitochondria and Chloroplast 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

Protein Transport into the Mitochondria and Chloroplast 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 the Mitochondria and Chloroplast Protein Transport into IM or IM Space Requires 2 Signal Sequences 1.Second signal =hydrophobic sequence; immediately after 1 st signal sequence 2.Cleavage of N-terminal sequence unmasks 2 nd 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 the Mitochondria and Chloroplast Protein Transport into Chloro Similar to Transport into Mito 1. occur posttranslationally 2. Use separate translocation complexes in ea membrane 3. Translocation occurs at contact sites 4. Requires energy and electrochemical gradient 5. Use amphilpathic N-terminal signal seq that is removed 6. Like the mito a second signal sequence required for translocation into thylakoid mem or space

Protein Transport into the Mitochondria and Chloroplast

Peroxisomes and Protein Import Peroxisomes ► Use O 2 and H 2 O 2 to carry out oxidative rxns ► Remove H from specific organic compounds RH 2 + O 2 R + H 2 O 2 ► Catalases use H 2 O 2 to oxidize other substances, particularly in liver and kidney detoxification H 2 O 2 + R’H 2 R’ + H 2 O ► Beta Oxidation ► Formation of plasmalogens (abundant class of phospholipids in myelin) ► Photorespiration and glyoxylate cycle in plants

Peroxisomes 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 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 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

ER and Protein Trafficking 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

ER and Protein Trafficking Quantity of SER and ER Dependent Upon Cell Type RER assoc. w/ protein synthesis SER assoc. lipid biosynthesis, detoxification, steroid synthesis, Ca 2+ storage

ER and Protein Trafficking Import of Proteins into ER ► Occurs co-translationally ► Signal recognition sequence recognized by SRP ► SRP recognized by SRP receptor ► Protein Translocator

ER and Protein Trafficking ► 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

ER and Protein Trafficking 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

ER and Protein Trafficking 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 Trafficking Signal Sequence is Removed from Soluble Proteins ► Two signaling functions: 1) directs protein to ER membrane 2) serves as “start transfer signal” to open pore ► Signal peptidase removes terminal ER signal sequence upon release of protein into the lumen

ER and Protein Trafficking Single Pass Transmembrane Proteins 1. N-terminal signal sequence initiates trans- location and additional hydrophobic “stop sequence anchors protein in membrane 2. Signal sequence is internal and remains in lipid bilayer after release from translocator 3. Internal signal sequence in opposite orientation 4. Orientation of start-transfer sequence governed by distribution of nearby chg aa

ER and Protein Trafficking Multipass Transmembrane Proteins ► Combinations of start- and stop-transfer signals determine topology ► Whether hydrophobic signal sequence is a start- or stop-transfer sequence depends upon its location in polypeptide chain ► All copies of same polypeptide have same orientation

ER and Protein Trafficking Folding of ER Resident Proteins ► ER resident proteins contain an ER retention signal of 4 specific aa at C- terminus ► PDI protein disulfide isomerase oxidizes free SH grps on cysteines to from disulfide bonds S-S allowing proteins to refold ► BiP chaperone proteins, pulls proteins posttranslationally into ER thru translocator and assists w/ protein folding

ER and Protein Trafficking Glycolsylation 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 as polypeptide enters lumen

ER and Protein Trafficking Oligosaccharide assembled sugar by sugar onto carrier lipid dolichol

ER and Protein Trafficking 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

ER and Protein Trafficking The Unfolded Protein Response

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 and Protein Trafficking Assembly of Lipid Bilayers on ER ► Scramblase phospholipid translocator equilibrates phospholipids distribution distribution ► Flipasses of PM responsible for asymmetric distribution of phospholipids

ER and Protein Trafficking 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

Transport of Molecules Btwn Nucleus and Cytosol