lecture 1 alt.pptx

Overview • Proteins are synthesized either on free ribosomes or on ribosomes bound to the membrane of endoplasmic reticulum (RER). • The synthesis of nuclear, mitochondrial and peroxisomal proteins occurs in the cytoplasm on free ribosomes. • Proteins that will eventually be associated with membrane...

Overview • Proteins are synthesized either on free ribosomes or on ribosomes bound to the membrane of endoplasmic reticulum (RER). • The synthesis of nuclear, mitochondrial and peroxisomal proteins occurs in the cytoplasm on free ribosomes. • Proteins that will eventually be associated with membranes (the endoplasmic reticulum, the Golgi complex and the plasma membrane) or secreted from the cell are synthesized on ribosomes that are bound to the endoplasmic reticulum, generating the rough ER (RER). Trafficking of proteins synthesized by ribosomes bound to ER • At the beginning of the translation process (protein synthesis), the new protein have a specific sequence (N-terminal) called signal sequence (N-terminal) • Signal sequence is recognized and bound to the Signal recognition particle (SRP) which will direct the ribosome to the ER . • The cytoplasmic side of ER contain receptors (SRP receptors) that specifically bind to SRP thus attaching the entire ribosome to the ER • SRP receptor is anchored (attached) to the translocation complex which is a kind of pore (hole) through it the protein pass into the lumen of ER as translation and elongation proceed. • As translation (protein synthesis)ended, the ribosomes dissociate (release) from the ER, the signal sequence is cleaved (removed) into the lumen and the protein makes its way to the correct membrane Protein modifications in the lumen of ER • while protein is getting synthesized, it will be modified by a process called as cotranslational process . • In the lumen (inside) of the ER, most of the proteins will be glycosylated (sugar coated):  A sugar will be transferred from the membrane lipid molecule (dolichol (lipid molecule): oligosaccharide (many sugars)) to the new protein. These new proteins contains either two of three amino acids (Asn-X-Ser and Asn-X-Thr) and the sugar will be fixed to the amide group (amino group) of Asn: N-glycosylation. Cytosol • The O-linked glycosylation occurs in the Golgi: carbohydrates (sugar) are attached to OH groups (hydroxyl group) of Ser or Thr amino acids within Asn-X-Ser or as Asn-X-Thr Proteins trafficking to Golgi • Some new proteins are used in ER, but most others start to move to Golgi through spaces within ER known as transitional element . • The ER membrane surround the nascent protein, buds off from the ER forming a transport vesicle.. • The transport vesicle transfer the new protein to Golgi complex, where it fuses with it • This fusion is mediated by proteins called v-SNARE, located on the surface of the vesicles V-SNARE vesicle SNARE T-SNARE target SNARE Golgi complex • The proteins from ER are sent to Golgi. It is the sorting organelle of the cell: “Post office of the cell”!! • As the proteins moved through the Golgi stacks, they are modified by specific Golgi enzyme. These modifications are important because they provide the signal that determine the final destination of the protein. e.g: Proteins destined to work in lysosomes are phophorylated at mannose (sugar) residues added to the protein, forming a mannose-6-P tag on lysosomal protein • The O-linked glycosylation occurs in the Golgi: carbohydrates (sugar) are attached to OH groups (hydroxyl group) of Ser or Thr amino acids within Asn-X-Ser or as Asn-X-Thr • Trans Golgi Network (TGN) is the final sorting and packing region of Golgi: Some new proteins will remain in Golgi and help in processing of new transported proteins, others, are send onward to lysosomes or to the outside of the cell. Lysosomes - Lysosomes are membrane enclosed organelle with acidic pH that contain enzymes called acid hydrolyases -Acid hydrolase enzyme are tagged by mannose-6-P in Golgi as marker showing their destination (lysosome) which are recognized by receptors for mannose 6P in certain regions of Trans Golgi Network (TGN) coated by clathrin protein. - New Mannose-6-P containing-proteins bind their receptors and the TGN membrane begins to bud off to enclose the new lysosomal proteins in a transport vesicle -Vesicles (destined for lysosomes) fuse with endosomes which are the transport vesicles from the plasma membrane created by endocytosis. The clathrin coat is lost .Clathrin is a protein that plays a major role in the formation of coated vesicles. - The pH is reduced within the precursor lysosome by the pumping in of protons (H+) and the new protein dissociate (released) from the M-6-P receptor and become functional enzymes - The receptors are recycled back to TGN. Secretion from the cell The are two other important processes that control the transportation of proteins after being sorted by the Trans Golgi Network specifically for the protein that is secreted outside the cell These processes are called constitutive and regulated secretion.  The constitutive secretion involves proteins that are important in maintaining cells and tissues, are constantly secreted [e.g., serum albumin and extracellular matrix (ECM)].  The regulated secretion occurs only when an external stimulus has been received, the vesicle containing the protein and cell membrane fuse on receiving the stimulus, and the protein is released outside the cell. This regulatory secretion pathway is used to secrete proteins such as digestive enzymes and hormones. Trafficking of proteins synthesized in free ribosomes • Proteins destined to remain in the cell and function in the cytosol, nucleus, mitochondria or peroxisomes are synthesized on free ribosomes. • Ribosomes synthesizing these proteins are free, because the protein lacks the N-terminal leading (signal) sequence that causes a ribosome to bind ER. • However, proteins are still tagged by special structures that direct them to organelle Cytoplasmic proteins • Are the proteins which remains in the cytosol outside organelles • They Lack leading signal and possess no structural feature that takes them to organelles Examples: enzymes of some pathways of carbohydrate metabolism e.g. glycolysis and glycogen metabolism Nuclear proteins • Most of these proteins that gain access to the nucleus are tagged by a nuclear localization signal (NLS) • They bind strongly to importin, a protein that (help) their nuclear entry through a nuclear pore. • The importin protein has alpha subunit and beta subunit and the nuclear protein with NLS bind to the alpha subunit. After that alpha importin binds with beta importin and collectively whole complex move inside nucleus • Then importin dissociate (a GTPdependent process (energy requiring )) and the new protein has reached its destination. Mitochondrial protein • The proteins are maintained in an unfolded form (non-functional form) prior (before) to entry in the mitochondria by the binding of chaperone proteins (that need ATP(energy) for their function). • These proteins are tagged by signal sequence called: import sequence present on N-terminal which bind to receptor located in Translocase of Outer Mitochondrial membrane (TOM) complex • • Once it reaches in the intermembrane space, the targeting signals binds to translocase of Inner Mitochondrial Membrane (TIM)TIM complex and the polypepetide chain enters the matrix. Once the protein is in the matrix, the signal sequence is removed from them and they fold properly and become functional. Transport into peroxisomes • Peroxisomes proteins are transported as Fully functional, fully folded proteins. • The proteins contain C-terminal tripeptide (SKL: SerLys-Leu) acting as peroxisomal targeting sequence(PTS). This sequence binds to a peroxisomal receptor which helps in entry of the protein into peroxisome. • • PTS is not cleaved after entry of peroxisome protein into peroxisome http://rsob.royalsocietypublishing.org