Protein sorting & targeting.pptx by SOMESH Govt. Nehru PG College

.pptx by SOMESH Govt. Nehru PG College Guided By Mr. AVINASH SINGH/Mr.PRABHAT BAIS

1. DEPARTMENT OF BOTANY SESSION 2023-24 PRESENTATION ON Protein sorting & targeting GUIDED BY Mr. AVINASH SINGH HEAD OF DEPARTMENT SUBMITTED BY SOMESH MSc BOTANY 2 ND Sem GOVT.NEHRU PG COLLEGE DONGARGARH

2. synopsis Introduction Central dogma What is protein targeting &translocation Targeting pathway Targeting signal Protein can move between different compartment Gated transport (Cytosol Nucleus) Targeting of Mitochondrial protein Targeting of chloroplast protein Targeting of peroxisomal protein Endomembrane system Targeting of protein in Endoplasmic reticulum Post translatoinal translocation in ER Five major modification in ER lumen Unfolded protein response Transport of protein from TGN to Lysosome Vesicle budding Vesicle trafficking & fusion

3. introduction The fate of protein synthesized by cytosolic (80s) ribosome depend on the present and absence of specific sorting signals that direct their transport from cytosol to specific orgenelle such as necleus , ER, Mitochondria, plastid, or peroxisomes . Without sorting signal remain in cytosol. Protein targeting or protein sorting is the mechanism by which a cell transport proteins to the appropriate position in the cell or outside of it . Protein that inter into the ER contain N- terminal ER sorting signal . Sorting signal are necessary for protein targeting .

4. Central dogma DNA synthesis maintain the genetic information and passes this to the next generation RNA synthesis (Transcription) is a transfer of the information from the DNA where it is stored into RNA which can be transported and interpreted. Ribosome translate the nucleotides on the mRNA into amino acid sequences producing a polypeptide.

5. transcription

6. translation

7. What is Protein targeting/translocation Both in prokaryote & Eukaryote newly synthesized proteins must be delivered to a specific subcellular location of exported from the cell for correct activity this phenomenon is called protein targeting . Protein targeting is necessary for proteins that are destined to work outside the cytoplasm. This delivery protein is carried out based on information contained in the protein itself . Fate of protein is decided by its amino acid sequence (sorting sequence). Each protein has its own sorting sequence. Signal peptide synthesis first at the time of protein synthesis. Signal peptide is on N terminus, then the specific cytosolic protein is recognized and taken to RER. After reaching of protein in the target organelle signal sequence are cleaved (except- Nucleus, peroxisome ).

8. Post translation translocation Even though most protein are co translationally translocated , some are translated in the cytosol and later transported to their destination . This occure for protein that go a mitochondrian , chloroplast, peroxisome,nucleus . Co-translational translocation Synthesized protein is transferred to an SRP receptor on the endoplasmic reticulum(ER) a membrane enclosed organelle. There, the nascent protein is inserted into the translocation complex (cytosol to ER) Post translational targeting (signal based targeting) Cytosol to Endoplasmic reticulum Nucleus Mitochondria Chloroplast Peroxixome Co translational targeting (vesicular based targeting) Start synthesis target on ER ER Golgy body Lysosome Plasma membrane Other secretory protein Cytosol

9. Targeting pathway

10. Targeting signals At the end of terminus N-terminus, C-terminus ( 15-60 amino long ) In the middle With sorting signal Without sorting signal Signal sequence Protein Signal patch Internal Terminal N-terminus C-terminus Cleavable Non-cleavable ( nc ) ( nc ) ( nc ) (Remain in cytosol)

11. Protein can move between compartments in different ways Cell to outside Cytosol to organelle Organelle to organelle Organelle to cytosol Gated translocation:- movement of protein from cytosol to nucleus by nuclear pore comlex Transmembrane transport:- C ytosol Endoplasmic reticulum Cytosol Mitochondria Cytosol Peroxisome Cytosol Chloroplast Vesicular transport:- Protein from ER to golgy apparatus and vesicle or protein cargo )

12. Endomembrane system It is 3D network of various vesicular system Present in all Eukaryote It is also called circulatory system of cell Endomembrane system comprises organelle:- Outer nuclear membrane Endoplasmic reticulum Golgy body Endosome Lysosome Single membrane bound organelle

13. Endoplasmic reticulum Largest single membrane bound intracellular compartment It extensive network of close 6 flatenned network Enclosed compartment called lumen Secretory protein or membran protein synthesis in ER bound ribosome & moved in lumen Five major modification in er lumen:- Addition of sugar molecule (N- linked glycosilation ) Formation of disulfied bond Proper folding Specific proteolytic cleavage Assembly into multimeric protein Function of SER:- lipid biosynthesis Site of the synthesis of steroid hormone Site of detoxification of various organic molecule ( alchohol in liver cell ) It is intracellular store house of calcium ion that regulate a variety of signaling pathway

14. Secretory pathway begins in er .

15. Targeting of protein in er :- Protein targeting to er lumen Protein that corporated into er membrane Protein targeted to ER lumen:- Protein synthesis begins in cytosol A protein that destined for secretion are target to ER membrane by a signal sequence (16-30 amino ) Signal sequence have one or more positevely charged amino acid adjacent to a sort stretch of 6-12 hydrophobic amino acid(hydrophobic patch) . ER signal sequence are located at the N-terminus(N-terminal hydrophobic signal sequence) Er protein targeting in Co-translational translocation microsome microsome microsome microsome microsome microsome Fig:- cell free system(co translation)

16. Protein targeting in er lumen SRP : - i t is cytosolic ribonucleoprotein SRP bind to the signal sequence & large ribosomal subunit direct to this complex to ER membrane. SRP bind to the signal sequence slow translation, a phenomenon termed elongation arrest P-54 subunit of SRP bind to the signal sequence SRP receptor It is integral membrane protein in the ER membrane Made up of a large α subunit & small β subunit Both SRP & SRP receptor have GTP binding site When SRP & SRP receptor bind, GTP hydrolysis capasity activate GTP hydrolysis gives power to bind Ribosome from Translocon α β SRP receptor

17. Post translation translocation In yeast some secretory protein translocated to ER lumen after the translation. SRP,SRP receptor not involve in post translational translocation . Interaction between signal sequence & translocon is sufficient for protein targeting Sec 61 act as translocon . Sec 63 complex tetrameric ( embedded in the ER membrane in vicinity to translocon ). Bip - it is the member of Hsp 70 family chaperon(help newly formed protein to its native conformation ). Bip present in ER lumen that have Polypeptide bindig site and ATPase activity. Sec 63 Signal peptidase Sec 63 ATP Bip ATP Bip Pi ADP Bip Sec 63 ADP Bip ADP Bip ATP Bip Pi

18. SIGNIFICATION OF GLYCOSILATION:- Proper folding in ER Provide stability(protease resistance) Cell cell interaction, adhesion. Oligosaccharide itself act as antigen.

19. Formation of disulfide bond Proper folding Formed of linkage group of two sulfhydry group (-SH) also known as thiol group present in two cystein residues. Tertiary & quartory of protein stablized . Disulfide bond only on secretory protein There are two type:- 1. intramolecular 2. intremolecular Calnexin :- In ER membrane, carbohydrate binding protein. Calreticulin :-in lumen protein prevent the further agregation of protein GLYCOSYLTRANSFERASE :- attach one molecule of glucose on nascent oligosacharide Nascent polypeptide calnexin calreticulin Bip PDI Unfolded protein response:- Ire 1:-integral membrane protein of ER. exist in monomeric (inactive) & dimeric (active) form.in dimeric form increase the production of Hac 1 Hac 1:- it is transcription factor that increase the transcription of protein folding catalyst

20. Vesicle type Transport mediated step Coat protein Associated GTPase COP II ER to GB(ANTEROGATE) Sec 23/Sec 24 & Sec13/31 Sec 16 Sar 1 COP I Cis Golgy to ER Later to early GB Coolomers of seven different Cop subfamily ARF CLATHRIN & ADAPTER PROTEIN Trans golgy to endosome PM to Endosome TGN to Endosome TGN to Lysosome , platelets vesicle Clathrin+AP 1 Clathrin+ GGA Clathrin+AP2 AP3 ARF

21. ECM (M6P) O-linked gylcosilation

22. Vesicular traffic

23. Transport of protein from the TGN to lysosome M6P bind to its receptor (pH 6.6-7.0) primarily found in TGN clathrin /AP1 coated vesicle bud from trans golgy network. Internal pH of late Endosome is (5.5) M6P leave the lysosomal enzyme Recognition determinants (signal patches) specific for lysosomal protein that recognized lysosomal protein & their Phosphyrilation

24. Vesicle budding & fusion The budding of vesicle initiate to polymerization of coat protein. Monomeric G protein (Arf1, Sar 1) play important role for coat protein polymerization 1 . Arf (help to budding of COP I or Clathrin coated vesicle 2. Sar I (help to COP II coated) Rab monomeric Gprotein :- help for vesicle fusion v SNAR,tSNAR :- contain all vesicle, use for budding & fusion GTPase activity switch Sar 1 GDP Sec 12 (GEF) Sar 1 GTP Sar 1 GTP Sar 1 GTP Sar 1 GDP Coat protein GDP-> GTP

25. microtubule

26. Gated transport:- cytosol to nucleus (nuclear protein)

27. TOM(outer membrane) TIM(inner membrane) MITOCHONDRIA IMPORT PROTEIN TOM (made up of 7 different protein) TIM(made up of 2 complex) TOM 20,22,70 (general import receptor) TOM 40 (general import translocon ) TOM5,6,7 (assembly of TOM complex) TIM 23 complex TIM 22 complex TIM 23,17 ( translocon inner membrane) TIM 50 (receptor) TIM 22 TIM 18 TIM 54 (involve in incorporation of hydrophobic domain of protein in inner membrane) Transmembrane transport cytosol to Mitochondria

28. Transmembrane transport TOM & TIM mediated OXA translocase mediated Imported by TOM &TIM22 complex Imported by TOM & SAM Imported by TOM & MIA Imported by TOM & MIM Imported by TOM & TIM23 complex cytosol to Mitochondria

29. targeting of choroplast protein Small subunit of Rubisco is sythesized by cytosolic ribosome Cytosolic Hsc 70 bind -> protein unfolded state Chloroplast that have N terminal stromal targeting sequence TOC complex:- translocase of the outer chloroplast membrane TIC complex:- translocase of the inner mitochondrial membrane protein are imported in unfolded state Cytosolic Hsc 70 ATP ADP Once protein reached in stroma N-terminal sequence has been cleaved Stromal Hsc 70 bind to the unfolded protein & Hydrolyzed ATP provide energy for translocation Hsp 60 help the unfolded polypeptide chain to fold.

30. Targeting of peroxisomal protein Serine,Lysine,lucine (S,K,L) amino acid or related sequence at the C-terminus are necessary for targeting Also called Peroxisomal targeting sequence ( PTS1 ) Protein targeted in folded form & spontaneous (no require energy). Pex 5 :- soluble( monomeric form) (bound dimeric form) receptor of PTS1 Pex 14 :- receptor of Pex 5 embeded in membrane Pex10,Pex12,Pex2 :- mono ubiquitinilation of Pex 5 Pex1,Pex6 :- interact with ubiquitinilated Pex5 with the help of Pex 15 & release it to cytosol. Pex 5 Pex 5 Pex 14 Pex 14 Pex 5 Pex 5 Pex 5 Pex 15 Pex 12 Pex 10 Pex 2 Pex 1 Pex 6 ub ub ub Pex 5 De- ubiquitinilation Pex 5

31. CONCLUSION Protein can be targeted to the inner space of an orgenelle different intracellular membrane , plasma membrane or to exterior of the cell via secretion . This delivery process is carried out based on information contain in the protein itself . correct sorting is crucial for the cell; error can lead to disease.

32. REFERENCE 1.pathfinde – usha mina,pranav kumar 2.principle of biochemistry- lehninger 3.lodish & baltimore

33. Thank you

34. SH SH C C S S - C C S S C C red PDI C-SH C-SH PDI C-SH C Oxi PDI C-S C-S