Protein targeting or translocation of proteins

Protein targeting or protein sorting is the mechanism by which a cell transports to the appropriate positions in the cell or outside of it. Both in prokaryotes and eukaryotes, newly synthesized proteins must be delivered to a specific sub-cellular 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 delivery process is carried out based on information contained in the protein itself. Correct sorting is crucial for the cell; errors can lead to diseases. 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.

1. Protein Targeting By Haider Ali Malik

2. Introduction Protein targeting Protein targeting or protein sorting is the mechanism by which a cell transports to the appropriate positions in the cell or outside of it. Both in prokaryotes and eukaryotes, newly synthesized proteins must be delivered to a specific sub-cellular location or exported from the cell for correct activity. This phenomenon is called protein targeting.

3. CENTRAL DOGMA 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. PROTEIN TARGETING Protein targeting is necessary for proteins that are destined to work outside the cytoplasm. This delivery process is carried out based on information contained in the protein itself. Correct sorting is crucial for the cell; errors can lead to diseases.

5. 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.

6. TARGETING PATHWAYS

7. 1- POSTTRANSLATIONAL TRANSLOCATION Posttranslational translocation is the pathway which occurs after the process of translation. 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.

8. 2- CO TRANSLATIONAL TRANSLOCATION In this pathway, transport of protein occurs during translation which is not completed fully. 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

9. TARGETING 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. In the absence of targeting signals, a protein will remain in the cytoplasm.

10. Types of targeting peptides 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 pre-sequence The internal targeting peptides

11. 1-PRESEQUENCES The pre-sequences of the targeting peptides are often found at the N-terminal extension but in case of peroxisomes the targeting sequence is on the C-terminal extension mostly. Signal sequence is a short peptide (usually 16-30 amino acids long) present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway. It is composed of between 6-136 basic and hydrophobic amino acids. Signal sequences are removed from the finished protein by specialized signal peptidases once the sorting process has been completed.

12. 2-INTERNAL TARGETING PEPTIDES The targeting peptides are often found at the with in polypeptide chain, not at any end .

13. Compartmental translocation of proteins There are three types of transport of proteins through different compartments of cell. Gated transport(Nucleus) Transmembrane transport(Mitochondria, Peroxisomes, chloroplast) Vesicular transport (E.R)

14. GATED TRANSPORT The protein transfer from or to nucleus is aided by nucleur pore. The nuclear pore complexes function as selective gates that actively transport (with expenditure of energy) specific macromolecules and macromolecular assemblies.

15. Protein targeting b/w cytosol and nucleus 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 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. 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 the cytosol.

16. IMPORT AND EXPORT OF PROTEINS TO NUCLEUS The transport is bidirectional and occurs through the nuclear pore complexes (NPCs). These are complex structures composed of aggregates of about 30 different proteins. The nuclear envelope has hundreds of NPCs, located where the two nuclear membranes meet. Each NPC has multiple copies of at least 30 different proteins called nucleoporins. Most polypeptides destined for the nucleus have address labels, called nuclear localization signals (NLSs), consisting of one or more short internal sequences with basic amino acids. Importins and Ran (a monomeric G‐protein that can exist in either the GTP‐bound or GDP‐bound conformation) help in import of proteins containing NLS. Proteins similar to importins, referred to as exportins , are involved in the export of many macromolecules (various proteins, tRNA molecules, ribosomal subunits and certain mRNA molecules) from the nucleus. Cargo molecules for export carry nuclear export signals (NESs). The family of importins and exportins are referred to as karyopherins .

17. TRANSMEMBRANE TRANSPORT Membrane-bound protein 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.

18. Protein Targeting to Mitochondria Protein translocation across mitochondrial membranes is mediated by multi-subunit protein complexes that function as protein translocators(TOM ,TIM 23,TIM22 ,OXA) TOM t ransports -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. OXA-mediates the insertion of inner membrane proteins .

19. PROTEIN TRANSPORT INTO THE MITOCHONDRIA Two distinct translocation complexes are situated in the outer and inner mitochondrial membranes, referred to as TOM (translocase-of-the-outer membrane) and TIM (translocase-of-the-inner membrane). Each complex is composed of a number of proteins, some of which act as receptors ( eg , Tom20/22 ) for the incoming proteins and others as components ( eg , Tom40 ) of the transmembrane pores through which these proteins must pass. Proteins must be in the unfolded state to pass through the complexes, and this is made possible by ATP dependent binding to several chaperone proteins including Hsp70. A proton-motive force across the inner membrane is required for import; it is made up of the electric potential across the membrane (inside negative) and the pH . The positively charged leader sequence may be helped through the membrane by the negative charge in the matrix. The pre-sequence is split off in the matrix by a matrix-processing protease (MPP).

20. Mit . Hsp70 ensures proper import into the matrix and prevents misfolding while interaction with the mtHsp60–Hsp10 system ensures proper folding. It is possible that the electric potential associated with the inner mitochondrial membrane causes a conformational change in the unfolded preprotein being translocated and that this helps to pull it across. Furthermore, the fact that the matrix is more negative than the intermembrane space may “attract” the positively charged amino terminal of the preprotein to enter the matrix. A number of proteins contain two signaling sequences —one to enter the mitochondrial matrix and the other to mediate subsequent relocation ( eg , into the inner membrane). Certain mitochondrial proteins do not contain pre-sequences ( eg , cytochrome c, which locates in the inter membrane space), and others contain internal pre-sequences. Overall, proteins employ a variety of mechanisms and routes to attain their final destinations in mitochondria.

21. Protein targeting to CHLOROPLAST In chloroplast, the targeting signal is correspondent to Transit peptide(TP ) 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. The signal sequence (transit peptide) binds with target protein along with chaperone cystolic Hsp70 . This is the signal to move the polypeptide through Toc complex. Stromal peptidase cleave the target sequence and pull the rest of polypeptide inside.

22. TRANSLOCATION OF PROTEIN IN CHLOROPLAST The vast majority of chloroplast proteins are synthesized as precursor proteins (preproteins) in the cytosol and are imported post-translationally into the organelle. Preproteins that contain a cleavable transit peptide are recognized in a GTP-regulated manner by receptors of 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 . The TOC and TIC translocons function together during the translocation process. 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.

23. VESICULAR TRANSPORT Proteins from the ER to the Golgi apparatus and proteins to E.R , for example, occurs in this way.

24. Protein Targeting in 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. The ER is organized into a netlike labyrinth of branching tubules and 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 cell exterior plus those destined for the lumen of ER, Golgi apparatus, or lysosomes are initially delivered to the ER lumen.

25. Protein Targeting in ENDOPLASMIC RETICULUM Most nascent proteins are transferred across the ER membrane into the lumen by the co-translational pathway, so called because the process occurs during ongoing protein synthesis. The process of elongation of the remaining portion of the protein being synthesized probably facilitates passage of the nascent protein across the lipid bilayer. It is important that proteins be kept in an unfolded state prior to entering the conducting channel—otherwise, they may not be able to gain access to the channel.

26. The signal sequence emerges from the ribosome and binds to the signal recognition particle (SRP). The SRP-ribosome-nascent protein complex travels to the ER membrane, where it binds to the SRP receptor (SRP-R). The SRP guides the complex to the SRP-R, which prevents premature expulsion of the growing polypeptide into the cytosol. The SRP is released, translation resumes, the ribosome binds to the translocon (Sec 61 complex), and the signal peptide inserts into the channel in the translocon . SRP and both subunits of the SRP-R can bind GTP which must be in the GTP form in both complexes to enable them to interact. When interaction occurs, GTP is hydrolyzed, SRP dissociates from SRP-R and is released, and the ribosome binds to the translocon , allowing the signal peptide to enter it. The signal peptide induces opening of the channel in the translocon by binding to certain hydrophobic residues in it, thus causing the plug. Protein Targeting in ENDOPLASMIC RETICULUM

27. Protein Targeting in ENDOPLASMIC RETICULUM The growing polypeptide is then fully translocated across the membrane, driven by its ongoing synthesis. The translocon consists of three membrane proteins (the Sec61 complex) that form a protein-conducting channel in the ER membrane through which the newly synthesized protein may pass. The channel opens only when a signal peptide is present, preserving conductance across the ER membrane when it closes. Closure of the channel when proteins are not being translocated prevents ions such as calcium and other molecules leaking through it, and causing cell dysfunction. Cleavage of the signal peptide by signal peptidase occurs, and the fully translocated polypeptide/protein is released into the lumen of the ER. The signal peptide is presumably degraded by proteases.

28. Protein Targeting in ENDOPLASMIC RETICULUM Ribosomes are released from the ER membrane and dissociate into their two types of subunits. Secretory proteins and soluble proteins destined for organelles distal to the ER completely traverse the membrane bilayer and are discharged into the lumen of the ER. Many secretory proteins are N-glycosylated. N-Glycan chains, if present, are added by the enzyme oligosaccharide protein transferase as these proteins traverse the inner part of the ER membrane — a process called co-translational glycosylation. 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 as to facilitate its recruitment into budding COPI-coated vesicles. (B) The retrieval of ER proteins begins in vesicular tubular clusters and continues from all parts of the Golgi apparatus.

29. TARGETING OF SECRETARY PROTEINS

30. SUMMARY 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.

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