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Lecture 4

Lecture 4 - Protein Synthesis and Transport

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Department
Biology
Course
Biology 2382B
Professor
Sashko Damjanovski
Semester
Winter

Description
LECTURE 4: PROTEIN SYNTHESISAND TRANSPORT Protein Sorting / Targeting • Typical mammalian cell: o 10,000 proteins o Must be localized correctly • Newly made peptides must be directed to the correct destination • Targeting: o Direct proteins to the right destinations (organelles) o During or after synthesis o Achieved by specific signal sequence of amino acids that help direct proteins to organelles • Sorting: o Direct proteins to the secretory pathway (ER, Golgi, lysosomes) General principles of protein synthesis, targeting and sorting • Many proteins are synthesized just by cytosolic ribosomes: o Those which remain in the cytosol o Those which are targeted to intracellular organelles such as (ER), mitochondria, chloroplasts, peroxisomes, and nucleus (they have a specific signal sequence) • Other proteins are synthesized by ribosomes attached to ER (the rough ER): o Those which reside in ER and proteins which are sorted to PM, Golgi complex, and lysosomes. • Accordingly, two major protein-sorting pathways are known: nonsecretory and secretory. • Secretory pathway – involves transport of proteins from the ER to their final destination within membrane-enclosed vesicles (generally to the outside of cell) • All nuclear-encoded mRNAs are translated on cytosolic ribosomes • Ribosomes synthesizing nascent proteins in the secretory pathway are directed to the RER by an ER signal sequence (steps 1, 2) • After translation is completed on the ER, these proteins can move via transport vesicles to the Golgi complex (step 3) • Further sorting delivers proteins either to plasma membrane or to lysosomes (step 4a, 4b) • mRNAentering the cytoplasm is recognized by ribosomes and other protein translational apparatus • Two options – can complete full synthesis of proteins or it can have a targeting sequence that acts as a flag for other proteins to transport it to organelles • ER signal sequences – recognized by certain proteins which will help guide newly emerging protein to the ER where it will undergo further processing • Important to understand that there are more ways – most start off with recognition by ribosomes and are translated; some have specific targeting sequences which are recognized by other proteins that help transport it to specific organelles; some proteins go through secretory pathways which are recognized by other proteins that guide it to the ER where it undergoes various modifications ER Structure • Uninterrupted membranous tubules and vesicles separated from cytoplasm • RER has ribosomes on the tubules (cisterna) • Cisterna are stacked • ER extend from nuclear membrane and starts to branch out in tubular system (cisterna) in stacked fashion Stringing the Concepts Together • Identify cellular features by microscopy, isolate & homogenize them to free organelles • Sucrose density-gradient centrifugation of homogenate allows for isolation of microsomes & ribosomes Secretory • SDS-PAGE is used to identify newly translated proteins proteins enter ER Functions • Secreted and membrane proteins are sorted through RER the ER • Sugars/carbohydrates are added to the polypeptide • Disulfide bonds are formed • Proteins are folded by helper proteins called chaperones Translocation and translation occur simultaneously Translation and translocation occur simultaneously. Cell-free experiments demonstrated that translocation of secretory proteins int2+microsomes is coupled to translation. Treatment of microsomes with EDTA, which chelates Mg ions, strips them of associated ribosomes, allowing isolation of ribosome-free microsomes, which are equivalent to ER membranes. Protein synthesis is carried out in a cell-free system containing functional ribosomes, tRNAs,ATP, GTP, and cytosolic enzymes to which mRNAencoding a secretory protein is added. The secretory protein is synthesized in the absence of microsomes (a) but is translocated across the vesicle membrane and loses its signal sequence (resulting in a decrease in molecular weight) only if microsomes are present during protein synthesis (b). RER: What are the major players? i)Amino terminal signal sequence of newly initiated polypeptide (nascent proteins) ii) Signal-Recognition Particle (SRP) – recognizes signal sequence iii) SRP receptor embedded in ER membrane – recognizes SRP iv) Translocon: protein channel – recognizes SRP receptor v) Cleavage site where signal sequence is cut by a signal peptidase Cotranslational Translocation • Steps 1, 2: once the ER signal sequence emerges from the ribosome, it Is bound by a SRP that helps direct it towards the ER membrane • Step 3: the SRP delivers the ribosome/nascent polypeptide complex to the SRP receptor in the ER membrane o This interaction is strengthened by binding of GTP to both the SRP and its receptor (increases affinity of receptor for SRP) • Step 4: transfer of the ribosome/nascent polypeptide to translocon leads to  ening of this translocation channel and insertion of the signal sequence and adjacent segment of the growing polypeptide into the central pore. o Both the SRP and SRP receptor, once dissociated from the translocon, hydrolyze their bound GTP (to GDP) and then are ready to initiate the insertion of another polypeptide chain • Step 5: as the polypeptide chain elongates, it passes through the translocon channel into the ER lumen, where the signal sequence is cleaved by signal peptidase and is rapidly degraded • Step 6: peptide chain continues to elongate as mRNAis translated toward the 3’end. Because the ribosome is attached to the translocon, growing chain is extruded through the translocon into the ER lumen • Steps 7, 8: once translation is complete, the ribosome is released, the remainder of protein is drawn into the ER lumen, the translocon closes and the protein assumes its native folded conformation Protein Modifications in ER • Specific proteolytic cleavage • Glycosylation • Formation of disulfide bonds • Folding of polypeptide chains Glycosylation in ER • Enzymatic transfer of 14-residue oligosaccharide precursor fro
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