Protein sorting

Cell Biology

1. Protein Sorting By  Mrs Sanchita Choubey (M.Sc., PGDCR, Pursuing Ph. D) Assistant Professor  of Microbiology Dr. D Y Patil Arts Commerce and Science College Pimpri , Pune

2. Introduction: Protein targeting Prot e in tar g et i ng o r pr o tein sorti n g i s the mec h anism b y w h i c h a ce l l transports proteins to the appropriate positions in the cell or outside of it.

3. T HE C ENTRAL D OGMA DNA synthesis maintains 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. Ribosomes translate the nucleotides on the mRNA into amino acid sequences producing a polypeptide

4. T RANSCRIPTION INITIATION E L ONG A T I O N TERMINATION

5. T RANSLATION Initiation – the assembly of a ribosome on an mRNA molecule. Elongation – repeated cycles of amino acid addition. Termination – the release of the new protein chain.

6. P ROTEIN TARGETING Both in prokaryotes and eukaryotes, newly synthesized proteins must be delivered to a specific subcellular location or 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 d eliv e ry p roc e ss i s ca r ried o u t based o n inf o rmat i o n contained in the protein itself. Co r rect sorting i s c r u cial f o r the c ell; err o rs can lead to diseases.

7. P ROTEIN TRANSLOCATION In 1970, Günter Blobel conducted experiments on the translocation of proteins across membranes. He was awarded the 1999 Nobel Prize for his findings. He discovered that many proteins have a signal sequence , that is, a short amino acid sequence at one end that functions like a postal code for the target organelle.

8. TARGETING PATHWAYS

9. P OSTTRANSLATIONAL TRANSLOCATION Even though most proteins are co translationally translocated, some are translated in the cytosol and later transported to their destination. This occurs for proteins that go to a mitochondrion , a chloroplast , or a peroxisome

10. C O TRANSLATIONAL TRANSLOCATION Synthisised 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

11. T ARGETING SIGNALS Targeting signals are the pieces of information that enable the cellular transport machinery to correctly position a protein inside or outside the cell. This information is contained in the polypeptide chain or in the folded protein. I n the a bse n ce of t arg e t i ng sig n als, a pro t ein w i l l rem a in i n the cytoplasm The continuous stretch of amino acid residues in the chain that enables targeting are called signal peptides or targeting peptides . There are two types of targeting peptides. The presequences and The internal targeting peptides

12. T HE P RESEQUENCES Th e pres e quences o f the tar geti n g p eptides are often found at the N-terminal extension . I t i s co m posed o f betw e en 6 - 13 6 b asic and hydrophobic amino acids. In case o f perox i so m es the tar g eting seq u ence is on the C-terminal extension mostly. signal sequences are removed from the finished protein by specialized signal peptidases once the sorting process has been completed

13. T HE INTERNAL TARGETING PEPTIDES the targeting peptides are often found at the with in polypeptide chain, not at any end .

14. P ROTEINS C AN M OVE B ETWEEN C OMPARTMENTS IN D IFFERENT W AYS Gated transport(Nucleus ) Transmembrane transport(Mitochondria, Peroxisomes,) Vesicular transport (E.R)

15. G ATED TRANSPORT The protein traffic between the cytosol and nucleus occurs between topologically equivalent spaces, which are in continuity through the nuclear pore complexes . The n u c l ear p o re c o mplexes fun c ti o n as selective gat e s that actively tr a ns p ort specific m a cro m olecules a nd macromolecular assemblies ,

16. T RANSMEMBRANE TRANSPORT M e m b ran e - bound prote i n translocators directly transport specific proteins across a membrane from the cytosol into a space that is topologically distinct. The transported protein molecule usually must unfold to snake through the translocator . The initial transport of selected proteins from the cytosol into the ER lumen or from the cytosol into mitochondria.

17. VESICULAR TRANSPORT Proteins from the ER to the Golgi apparatus and proteins to E.R , for example, occurs in this way. transport intermediates— which may be small, spherical transport vesicles or larger, irregularly shaped organelle fragments— ferry proteins from one compartment to another. The transfer of soluble recognized by a complementary receptor in the appropriate membrane.

18. G ATED TRANSPORT

19. THE TRANSPORT OF MOLECULES BETWEEN THE NUCLEUS AND THE CYTOSOL The nuclear envelope encloses the DNA and defines the nuclear compartment. This envelope consists of two concentric membranes that are penetrated by nuclear pore complexes. The inner and outer nuclear membranes are continuous, they maintain distinct protein compositions. The inner nuclear membrane contains specific proteins that act as binding sites for chromatin and for the protein meshwork of the nuclear lamina that provides structural support for this membrane. The inner membrane is surrounded by the outer nuclear membrane, which is continuous with the membrane of the ER. Like the membrane of the ER the outer nuclear membrane is studded with ribosomes engaged in protein synthesis . The proteins made on these ribosomes are transported into the space between the inner and outer nuclear membranes (the perinuclear space), which is continuous with the ER lumen. with ribosomes engaged in protein synthesis. Bidirectional traffic occurs continuously between the cytosol and the nucleus. The many proteins , histones, DNA and RNA polymerases, gene regulatory imported into the nuclear compartment from the cytosol, proteins, and RNA-processing proteins are selectively tRNAs and mRNAs are synthesized in the nuclear compartment and then exported to t he cytosol

20. I M PO R T A N D EX P O R T OF P ROTEINS TO NUCLEUS Protein encodes a receptor protein that is specialized for the transport of a group of nuclear proteins sharing structurally similar nuclear localization signals. Nuclear import receptors do not always bind to nuclear proteins directly. Additional adaptor proteins are sometimes used that bridge between the import receptors and the nuclear localization signals on the proteins to be transported. Export -ribosomal subunits and RNA molecules. For import and export requires energy

21. T RANSMEMBRANE TRANSPORT

22. M I T OCHON D RIA A N D C H LOROPL A STS Mitochondria and chloroplasts are double- membrane-enclosed organelles. They specialize in the synthesis of ATP , using energy derived from electron transport and oxidative phosphorylation in mitochondria and from photosynthesis in chloroplasts . Both organelles contain their own DNA, ribosomes , and other components required for protein synthesis . Their growth depends mainly on the import of proteins from the cytosol.

23. THE TRANSPORT OF PROTEINS INTO MI T OC H ON D RIA A N D C H LO R OPL A STS

24. Protein translocation across mitochondrial membranes is mediated by multi-subunit protein complexes that function as protein translocators. TOM ,TIM 23,TIM22 ,OXA TOM transports -mitochondrial precursor proteins , nucleus- encoded mitochondrial proteins. TIM23-proteins into the matrix space. TIM22 -mediates the insertion of a subclass of inner membrane proteins, including the carrier protein that transports ADP, ATP, and phosphate. O X A - mediat e s the i n sertion of inner membrane proteins .

25. P ROTEIN T RANSPORT INTO THE M ITOCHONDRIA Import of Mitochondrial Proteins Post-translational: Unfolded polypeptide chain precursor proteins bind to receptor proteins of TOM 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

26. P ROTEIN T RANSPORT INTO THE M ITOCHONDRIA Import of Mitochondrial Proteins Requires ene r gy in f o rm of A TP a nd H+ gradient and a s sitan c e of hsp70 -re l e a se of unfold e d prot e ins from hsp70 requir e s A TP h y dro l y sis -once thru TOM and bound to TIM, translocation thru TIM requires electrochemical gradient

27. P ROTEIN T RANSPORT INTO THE M ITOCHONDRIA Protein Transport into IM or IM Space Requires 2 Signal Sequences Second signal =hydrophobic sequence; immediately after 1 st signal sequence Cleavage of N-terminal sequence unmasks 2 nd signal used to translocate protein from matrix into or across IM using OXA OXA also used to transport proteins encoded in mito into IM Alternative route bypasses matrix; hydrophobic signal sequence = “stop transfer”

28. C HLOROPLAST The preprotein for chloroplasts may contain a stromal import sequence or a stromal and thylakoid targeting sequence. The majority of preproteins are translocated through the Toc and Tic complexes located within the chloroplast envelope. In the stroma the stromal import sequence is cleaved off and folding as well as intra- chloroplast sorting to thylakoids continues. Proteins targeted to the envelope of chloroplasts usually lack cleavable sorting sequence.

29. TRANSLOCATION OF PROTEIN IN CHLOROPLAST The vast majority of chloroplast proteins are synthesized as precursor proteins (preproteins) i n t he c y to s ol a n d are i mp ort e d p o s t- translationally into the organelle. Most proteins that are destined for the thylakoid membrane, Preproteins that contain a cleavable transit peptide are recognized in a GTP-regulated manner12 by receptorsof the outer-envelope translocon, which is called theTOC complex. The preproteins cross the outer envelope through an aqueous pore and are then transferred to the translocon in the inner envelope,which is called the TIC complex . T h e T OC a n d T IC together during the tra ns l o co n s tra ns l o c a ti o n f un ct io n pro c e ss Completion of import requires energy,which probably comes from the ATP-dependent functioning of molecular chaperones in the stroma. The stromal processing peptidase then cleaves the transit sequence to produce the mature form of the protein, which can fold into its native form.

30. THE ENDOPLASMIC RETICULUM All eukaryotic cells have an endoplasmic reticulum (ER). Its membrane typically constitutes more than half of the total membrane of an average animal cell. T he E R i s or g anized in t o a net l i ke labyr i nth of branch i ng tu b ul e s a nd flattened sacs extending throughout the cytosol , to interconnect. The ER has a central role in lipid and protein biosynthesis . Its membrane is the site of production of all the transmembrane proteins and lipids for most of the cell’s organelles ( the ER itself, the Golgi apparatus, lysosomes, endosomes, secretory vesicles, and the plasma membrane). The ER membrane makes a major contribution to mitochondrial and peroxisomal membranes by producing most of their lipids. almost all of the proteins that will be secreted to the cell exterior plus those destined for the lumen of the ER, Golgi apparatus, or lysosomes are initially delivered to the ER lumen.

31. TRANSLOCATION OF PROTIENS IN E.R

32. VESICULAR TRANSPORT

33. U TILIZATION OF DIFFERENT COATS IN VESICULAR TRAFFIC

34. VESICULAR TRANSPORT FROM ER TO GC

35. Those ER resident proteins that escape from the ER are returned to the ER by vesicular transport. (A) The KDEL receptor present in vesicular tubular clusters and the Golgi apparatus, captures the soluble ER resident proteins and carries them in COPI- coated transport vesicles back to the ER. Upon binding its ligands in this low-pH environment, the KDEL receptor may change conformation, so a s to into faci l ita t e its recr u it m e n t b u d d i n g COPI- co a ted vesicles. (B) The retrieval of ER proteins begins in vesicular tubular clusters and continues from all parts of the Golgi apparatus.

36. TARGETING OF SECRETARY PROTEINS

37. T HE G OLGI APPARATUS The Golgi apparatus is integral in modifying, sorting, and packaging these macromolecules for cell secretion ( exocytosis ) or use within the cell. Post office; it packages and labels items(a mannose-6-phosphate label to proteins destined for lysosomes ) which it then sends to different parts of the cell. glycosylation refers to the enzymatic pro c ess that attaches glycans to proteins , lipids , or other organic molecules . a fo r m of c o- post-translational Glycosylation is tr a n s lat i o n al a n d modification

38. Five classes of glycans are produced : N-linked glycans attached to nitrogen of asparagine or arginine side- chains. N-linked glycosylation requires participation of a special lipid called dolichol phosphate. O-linked glycans attached to the hydroxy oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side-chains, or to oxygens on lipids such as ceramide phospho-glycans linked through the phosphate of a phospho-serine; C-linked glycans, a rare form of glycosylation where a sugar is added to a carbon on a tryptophan side-chain Glypiation, which is the addition of a GPI anchor that links proteins to lipids through glycan linkages.

39. P ROTEIN TRAFFICKING OR SITE SPECIFIC TRANSPORT

40. S UM M A R Y Both in prokaryotes and eukaryotes, newly synthesized proteins must be delivered to a specific subcellular location or exported from the cell for correct activity. This phenomenon is called protein targeting. Secretory proteins have an N-terminal signal peptide which targets the protein to be synthesized on the rough endoplasmic reticulum (RER). During synthesis it is translocated through the RER membrane into the lumen. Vesicles then bud off from the RER and carry the protein to the Golgi complex, where it becomes glycosylated. Other vesicles then carry it to the plasma membrane. Fusion of these transport vesicles with the plasma membrane then releases the protein to the cell exterior.

41. R EFERENCES Molecular Biology, Third Edition ( Phil Turner, Alexander McLennan,Andy Bates & Mike White) Pal a d e G ( 197 5) I n t r a cell u lar aspec t s o f t h e process of protein synthesis. Science 189, 347–358. L o dis h , H. , B e r k , A. , Z i p u rsk y , S . L ., M a tsud a ir a , P . , Baltimore, D ., Darnell, J . , 2000 , Molecular C e ll B i o log y , Th i rd Edition ( D a v id Ha m es & Nig e l Bioche m istr y , Hooper, ) 4 th Ed., W.H. Freeman. http://bcs.whfreeman.com/lodish5e/ L e hninger pri n c ip l es o f B i och e mistr y , F o urth e d iti o n , David L. Nelson, Michael M. Co