Chapter 16-part II. Translocation into chloroplast occurs via a similar strategy to the one used by mitochondira

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Chapter 16-part II Translocation into chloroplast occurs via a similar strategy to the one used by mitochondira Both occur post-translationally Both use two translocation complexes, one at each

1 Chapter 16-part II Translocation into chloroplast occurs via a similar strategy to the one used by mitochondira Both occur post-translationally Both use two translocation complexes, one at each membrane Both require energy Both remove the signal sequence after transfer However chloroplasts have a H+ gradient across the thylakoid membrane and use GTP hydrolysis to drive transfer Multiple signals and pathways target proteins to submitochondrial compartments Matrix targeting Target: 1.inner-membrane 2.Intermembrane- space 3.Out-membrane: unknow mechanism 4.matrix

membrane has three pathway A,B,C Inner-membrane proteins: three separate pathways (B)

2 Outer-membrane proteins Inner-membrane proteins: three separate pathways (A) Short matrix-targeting sequence is followed by long stretch of hydrophobic amino acids Inner membrane has three pathway A,B,C Inner-membrane proteins: three separate pathways (B) Inner-membrane proteins: three separate pathways (C) No N-terminal matrix-targeting sequences

Three pathways for targeting inner-membrane proteins A B C 1. N-terminal matrix 1.

Hydrophobic stop- Enter & redirect membrane translocation transfer anchor sequence (not clear) channel 4. Lateral movement 3.

Lateral movement Oxa1 also participates in the inner-membrane insertion of certain proteins encoded by mitochodrial DNA

intermembrane space (intermembrane-space proteins) Two pathway for transporting proteins from the cytosol to the mitochondrial

3 Three pathways for targeting inner-membrane proteins A B C 1. N-terminal matrix 1. Multiple internal targeting sequence sequences 2. Cleave 1st sequence 2. Use different inner 3. Hydrophobic stop- Enter & redirect membrane translocation transfer anchor sequence (not clear) channel 4. Lateral movement 3. Hydrophobic stoptransfer anchor sequence 4. Lateral movement Oxa1 also participates in the inner-membrane insertion of certain proteins encoded by mitochodrial DNA synthesized in matrix by mitochondrial ribosomes Two pathway for transporting proteins from the cytosol to the mitochondrial intermembrane space (intermembrane-space proteins) Two pathway for transporting proteins from the cytosol to the mitochondrial intermembrane space 1. Two targeting sequences 2. First N-terminal matrix targeting sequence removed 3. Second sequence = hydrophobic stop-transfer anchor sequence stay in membrane 4. Protease cleaves; protein folds Intermembrane space targeting sequence Direct delivery to the inner membrane space

Mitochondrial porin (P70) N-terminal sequence is important P70 is hydrophobic, for stop-transfer, prevents

chloroplast stromal proteins is similar to import of mitochondrial martix protein Proteins are targeted to

Protein Synthesis in cytosol transport thylkaoid photosynthesis SRP: signal-recognition particle Have four

4 Similar to Fig Outer mitochondrial membrane Protein target to outer-membrane Unclear mechanisms Need energy Mitochondrial porin (P70) N-terminal sequence is important P70 is hydrophobic, for stop-transfer, prevents transfer of the protein into matrix and anchors protein Protein target to chloroplast Targeting of chloroplast stromal proteins is similar to import of mitochondrial martix protein Proteins are targeted to thylakoids by mechanisms related to translocation across the bacterial inner membrnae. Protein Synthesis in cytosol transport thylkaoid photosynthesis SRP: signal-recognition particle Have four types of transport protein to chloroplast: closely related to bacteria. Type I: SRP dependent Type II: related Sec A Type III: related mitochondrial Oxa 1 Type IV: for metal-containing protein, ph pathway Type I R: arginine Type IV

Type III Type II Chloroplast encoded protein transport into

membrane: Peroxisome activity: Peroxisomes are single membrane

Peroxisomes house a variety lipid oxidation reactions, these often

5 Type III Type II Chloroplast encoded protein transport into thylakoid membrane Fig16-23 Four routes across the thylakoid membrane: Peroxisome activity: Peroxisomes are single membrane organelles. Peroxisomes house a variety lipid oxidation reactions, these often utilize H 2 O 2 hydrogen peroxide and/or O2 (e.g. catalase & urate oxidase) Examples: B-oxidation & breakdown of fatty acids Detoxification of H 2 O 2 (catalase) 2 H 2 O 2 2H 2 O + O 2 Synthesis of certain phospholipids (e.g. plasmalogen) Cholesterol breakdown to bile acids

16.6 Sorting of peroxisomal proteins Peroxisomes are bounded by single membrane. No DNA and ribosomes, all protein encoded by nuclear gene.

the peroxisomal matrix. PTS1: Ser-LyS-Leu (SKL) at C-terminal, - not cleaved after internalization (very different) - translocate folded protein. Similar to SRP and SRP receptor transport pathway.

Perioxisomal targeting sequence soluble Synthesis and targeting of perioxisomal proteins Encoded by nuclear DNA Synthesized on free ribosomes Proteins are folded in the cytosol then transported

6 16.6 Sorting of peroxisomal proteins Peroxisomes are bounded by single membrane. No DNA and ribosomes, all protein encoded by nuclear gene. Has catalase for H2O2 into H2O, are most abundant in liver cell about 1-2% Transport protein into peroxisomes by cytolsolic receptor Pex5 targets protein with an SKL sequence at the C-terminus into the peroxisomal matrix. PTS1: Ser-LyS-Leu (SKL) at C-terminal, - not cleaved after internalization (very different) - translocate folded protein. Similar to SRP and SRP receptor transport pathway. Folded proteins can be translocated across the membrane Need ATP. Perioxisomal targeting sequence soluble Synthesis and targeting of perioxisomal proteins Encoded by nuclear DNA Synthesized on free ribosomes Proteins are folded in the cytosol then transported Perioxisomal targeting sequence (PTS1) Ser-Lys-Leu (SKL) PST1-tagged protein binds to soluble receptor in the cytosol Brought to the peroxisomal surface Moved through translocation channel (not clear) Soluble receptor dissociates Needs ATP for translocation PTS1 is not cleaved A short signal sequence directs the import of proteins into peroxisomes Peroxisomal membrane and matrix proteins are incorporated by different pathways Translocation channel protein Pex10, Pex12 and Pex2 are important. Any mutation of channel catalase can not enter peroxisome Catalase inner peroxisomes Pex12 No catalase Pex19 like receptor for membrane targeting Pex3 and Pex16 are membrane protein for transport Division need Pex 11 New peroxisomes are derived by growth and splitting of existing peroxisomes Fig Model of peroxisomal biogenesis and division

Zellweger syndrome Characterized by a variety of neurological, visual, and liver abnormalities leading to death during early infancy.

biogenesis. Zellweger syndrome is caused by the mistakes of protein import into the peroxisomes. Accumulation of long chain fatty acids in plasma and tissues.

Mitochondrion N-terminal Chloroplast N-terminal Peroxisome C-terminal Nucleus Internal How do proteins get to other organelles?

7 Zellweger syndrome Characterized by a variety of neurological, visual, and liver abnormalities leading to death during early infancy. Transport of proteins into peroxisomal matrix is impaired; Genetic analyses of different Zellweger patients & of yeasts carrying similar mutations identify >20 genes required for peroxisomal biogenesis. Zellweger syndrome is caused by the mistakes of protein import into the peroxisomes. Accumulation of long chain fatty acids in plasma and tissues. Causing severe impairment of many organs and death. Peroxisomal disorders affecting either peroxisomal biogenesis or transport into peroxisomes affect fatty acid and lipid metabolism. Mitochondrion N-terminal Chloroplast N-terminal Peroxisome C-terminal Nucleus Internal How do proteins get to other organelles? 3 5 nonconsecutive Arg or Lys residues, often with Ser and Thr; no Glu or Asp residues No common sequence motifs; generally rich in Ser, and Thr and small hydrophobic amino acids, poor in Glu and Asp residues Usually Ser-Lys-Leu at extreme C-terminus One cluster of 5 basic amino acids, or two smaller clusters of basic residues separated by 10 amino acids

Nuclear Cargo Imported Polymerases Histones Transcription factors Ribosomal proteins 10 6 ribos=>560k ribo proteins

etc. Exported trnas mrnps Ribosomal subunits Transcription factors 12.

509-514 In nucleus: DNA premrna binding hnrnp splicing m-rna export to cytosol via nuclear envelop translation From

called heterogenous nuclear nuclear RNA (hnrna) Mature mrna + associated specific hnrnp messenger ribonuclear

8 Nuclear Cargo Imported Polymerases Histones Transcription factors Ribosomal proteins 10 6 ribos=>560k ribo proteins imported/min 14,000 ribo subs exported/min 3-4K pores/cell=> 150 ribo proteins/min/pore Also 100 histones/min/pore etc. Exported trnas mrnps Ribosomal subunits Transcription factors 12.3 Macromolecular transport across the nuclear envelop pp In nucleus: DNA premrna binding hnrnp splicing m-rna export to cytosol via nuclear envelop translation From immature to mature mrna are associated with heterogeneous ribonucleoprotein particles (hnrnp); mrna + hnrnp also called heterogenous nuclear nuclear RNA (hnrna) Mature mrna + associated specific hnrnp messenger ribonuclear protein complex; mrnp Fig 12-1 Nascent transcripts are associated with hnrnp proteins Protein components with RNA Recognition Motif (pre-mrna) hnrnps (heterogeneous ribonucleoprotein particles) nucleus cytosol Nuclear pore complex (NPC)

9 Figure 8.2 Electron Micrograph Showing Nuclear Pores Figure 8.6 Electron Micrograph of Nuclear Pore Complexes eightfold symmetry organized around a large central channel. Nuclear pore complex (NPC) Nuclear pore complexes perforate the nuclear envelope hydorphilic Has FG (phenylalanine glycine) amino acids repeat (hydrophobic) Also called FG-nucleoporins < 60kD by water diffusion Large protein need other carrier or protein participate Composed by more than 50 different proteins called nucleoporins.

10 Nuclear pore complex (NPC) Elaborate ( 精密 ) structure of approx. 100 proteins forming protein lined aqueous channel approx 9nm diameter Protein fibrils protrude each side of complex - form cage-like structure on nuclear side, consist of nucleoporin (yeast 590 types, mammal 100types) Typical cell contains pore complexes Each pore, on average, imports 100 histone molecules per minute and exports 6 small ribosomal subunits. The formed protein also called nucleoporin Nuclear Pore Complexes (NPC) NPCs span the inner and outer nuclear membrane in typical mammalian cell nucleus. NPC are: large million Da complex- composed of more than 50 different proteins gated - Have diffusion limit of kda. Larger proteins require active transport busy - every minute each NPC must transport 100 histone proteins, 6 small and large ribosome subunits, plus numerous other proteins and RNP complexes. traffic is bi-directional and highly regulated. Two mechanisms for nuclear/cytoplasmic transport small molecular weight molecules can pass by diffusion (energy independent) < 5000 daltons - freely permeable ~17,000 daltons - 2 min equilibration > 60,000 daltons- impermeable to nuclear entry. But what about ribosomal subunits, mrnp particles with molecular weights in millions? Transport through NPCs Import (chromatin proteins, ribosomal proteins RNA processing proteins, proteins that shuttle between nucleus and cytoplasm) players: nuclear import receptors The GTPase Ran and its regulators Sequence on protein to be imported -Nuclear localization signal (NLS) Export (ribosome subunits, mrna complexes, trnas, shuttling proteins ) players: nuclear export receptors Ran and its regulators Sequence on protein to be exported - Nuclear export signal (NES)

Nucleus imports and exports macromolecules 26nm 9 nm = diffusion limit 26 nm = active transport also accumulation versus diffusion 9nm 15nm Nuclear envelope encloses nuclear DNA

and ribosomal subunits are exported out of nucleus Nuclear envelope perforated by pores, movement occurs in both directions through these pores Mechanism of protein import

11 Nucleus imports and exports macromolecules 26nm 9 nm = diffusion limit 26 nm = active transport also accumulation versus diffusion 9nm 15nm Nuclear envelope encloses nuclear DNA Inner membrane contains binding sites for chromosomes and nuclear lamina Outer nuclear membrane resembles ER membrane Transcription factors enter into nucleus, RNA (once spliced) and ribosomal subunits are exported out of nucleus Nuclear envelope perforated by pores, movement occurs in both directions through these pores Mechanism of protein import Proteins selected for import into nucleus have a nuclear localisation signal (NLS) e.g. -Pro-Pro-Lys-Lys-Lys-Arg- Position of NLS in protein not important except needs to be on surface Nuclear localization signals (NLS) direct nuclear proteins to the nucleus nucleus Protein enter nucleus NLS is recognized by cytosolic nuclear import receptors which bind to nuclear pore fibrils extending into cytosol Pore opens and protein plus import receptor enter nucleus. Import receptor exported for re-use Protein can not enter nucleus Keep in cytosol

Importins transport protein containing Nuclear-Localizing Signal into the nucleus Colloidal gold spheres coated with peptides containing NLS Nuclear pore transport (large

Cytosol ER translation to protein contain NLS bind to importin import nucleus From digitonin-permeabilized cell system: Ran (G protein) NFT2 (nuclear transport factor 2)

function Nuclear import: example of a NLS Simian virus 40 large T antigen (SV40 T) NLS: PKKKRKV (single amino acid code) Pro-Lys-Lys-Lys-Arg-Lys-Val Pyruvate kinase is

12 Importins transport protein containing Nuclear-Localizing Signal into the nucleus Colloidal gold spheres coated with peptides containing NLS Nuclear pore transport (large aqueous pore) is fundmental different from organelle transport (lipid bilayer). Cytosol ER translation to protein contain NLS bind to importin import nucleus From digitonin-permeabilized cell system: Ran (G protein) NFT2 (nuclear transport factor 2) importin α and importin β formed heterodimeric nuclear-import receptor α bind to NLS (hydrophobic)of cargo; β bind to FGnucleoprin Some studies has found only β has import function Nuclear import: example of a NLS Simian virus 40 large T antigen (SV40 T) NLS: PKKKRKV (single amino acid code) Pro-Lys-Lys-Lys-Arg-Lys-Val Pyruvate kinase is cytoplasmic Pyruvate kinase + SV40 T NLS is nuclear Digitonin is detergent permeabilizes plasma membrane Synthetic SV40 T-antigen NLS observation: Lodish Fig High levels of SV40 T NLS competitively inhibits nuclear import of NLS-bearing proteins. Hypothesis: receptors required for nuclear import.

Nuclear import receptors bind nuclear localization signals (α subunit) and nucleoporins (β subunit) Nuclear export works like nuclear import, but in reverse Nuclear export signals & nuclear export

guanine nucleotide triphosphate (GTP) Ran exists in two conformational forms: Ran - GTP and Ran- GDP Ran has weak GTPase activity that is stimulated by RanGAP (GTPase activating protein) A guanine

hydrolysis, release provides energy for nuclear transport asymmetric localization of RanGAP and RCC1 assure directionality of transport Protein import to nucleus α+β Low affinity to NLS After import

13 Nuclear import receptors bind nuclear localization signals (α subunit) and nucleoporins (β subunit) Nuclear export works like nuclear import, but in reverse Nuclear export signals & nuclear export receptor & nuclear transport receptor (karypherins) trna or 5S RNA: nuclei cytosol NLS-particle: cytosol nuclei Active nuclear transport is driven by Ran Ran is a small GTPase- binds and hydrolyses guanine nucleotide triphosphate (GTP) Ran exists in two conformational forms: Ran - GTP and Ran- GDP Ran has weak GTPase activity that is stimulated by RanGAP (GTPase activating protein) A guanine nucleotide exchange factor (GEF), called RCC1 stimulates the release of GDP and binding of GTP (higher concentration of GTP in the cell assures preferential binding of GTP) The cycle of GTP binding, hydrolysis, release provides energy for nuclear transport asymmetric localization of RanGAP and RCC1 assure directionality of transport Protein import to nucleus α+β Low affinity to NLS After import to nucleoplasm high concentration export Transport without ATP Interact with FGnucleoprins GTPase accelerating protein Protein import through the nuclear pore complex. Proteins are transported through the nuclear pore complex in two steps. First the protein with a classical basic amino acid-rich nuclear localization sequence (NLS) is recognized by importin α, which forms a complex with importin β. Importin β binds to the cytoplasmic filaments of the nuclear pore complex, bringing the target protein to the nuclear pore. The protein and importin α are then translocated through the nuclear pore complex in a second, energy-requiring step, which requires GTP hydrolysis by the Ran protein.

Not all proteins that enter the nucleus have NLS sequences Importin beta structure Nuclear import do not always

Soluble cytosolic protein FG-repeat (Phe-Gly) serve as binding sites for the import receptors.

molecules RNA moves through nuclear pore as complex of ribonucleoprotein (RNP) Protein component of RNP contains

14 Not all proteins that enter the nucleus have NLS sequences Importin beta structure Nuclear import do not always bind to nuclear proteins directly. Soluble cytosolic protein FG-repeat (Phe-Gly) serve as binding sites for the import receptors. Mechanism of export from nucleus Most of traffic moving out of nucleus consists of different types of RNA molecules RNA moves through nuclear pore as complex of ribonucleoprotein (RNP) Protein component of RNP contains a nuclear export signal (NES) that is recognised by export proteins Ran = GTPase GAP = GTPase-activing protein GEF = Guanine exchange factor mrna is bound by hnrnp only after fully spliced so only mature RNA is exported

Heterokaryon assay demonstrating that human hnrnp A1 protein (red) can cycle in and

fusion heterokaryon (one cell contain many nucleus) add cycloheximide inhibited

From HeLa hnrnp1 transport to xenpous nucleus HeLa cell hnrnp1 hnrnpc Mechanism of

Exportins transport proteins containing NES out of the nucleus Nucleus export signal

15 Heterokaryon assay demonstrating that human hnrnp A1 protein (red) can cycle in and out of the cytoplasm but human hnrnp protein C (grn) cannot HeLa cell + Xenpous cell fusion heterokaryon (one cell contain many nucleus) add cycloheximide inhibited protein synthesis Xenpous Original: hnrnp only Specific binding for human HeLa cell From HeLa hnrnp1 transport to xenpous nucleus HeLa cell hnrnp1 hnrnpc Mechanism of transport through nuclear pores C: hnrnpa1 protein (nucleus cytosol nucleus) Exportins transport proteins containing NES out of the nucleus Nucleus export signal A least three type of NES: PKI (leucine rich), 38-residue in hnrnp A1,a squence in hnrnp k. Conformal change high affinity to NES Interact with FGnucleoprins hnrnp and mrna by the same mechanism Exportin-t : bind to trna and interact with Ran export trna to cytoplasm

16 FG-repeat proteins Bidirectional model The Ran GTPase drives directional transport through nuclear pore complexes

Chapter 12: Intracellular sorting

Chapter 12: Intracellular sorting Principles of intracellular sorting Principles of intracellular sorting Cells have many distinct compartments (What are they? What do they do?) Specific mechanisms are