CBS Protein targeting KEATS 22_23 acc.pdf

Faculty of Life Science and Medicine Stuart Knight Foundations of Medical Science Cell Biology and Signalling block Biochemistry Department Protein Targeting Teaching Objectives Describe the different w...

Faculty of Life Science and Medicine Stuart Knight Foundations of Medical Science Cell Biology and Signalling block Biochemistry Department Protein Targeting Teaching Objectives Describe the different ways in which proteins can be targeted to organelles or for secretion in eukaryotic cells Indicate the function of signal peptides and ‘postcode’ sequences in directing proteins into their appropriate locations Appreciate how defects in targeting can result in human disease 2  Protein targeting - outline Cellular function in compartmentalised – this can involve the plasma membrane e.g. cell polarity Function is defined by proteins Proteins have to be directed to different parts of cell – Organelles – Membrane proteins – Secreted proteins – Cytosolic proteins 3 Protein sequences act as “postcodes” to direct proteins to correct location SE23 1JU SE23 1NH  Clinical case We will be discussing a clinical case during this lecture – the details will be posted on KEATS after the lecture 5  Targeting of protein is encoded in its sequence Targeting proteins to ER via signal peptide – Secreted proteins – Membrane proteins – Lysosomal proteins Targeting cytosolic protein to organelles via other peptide signals – Mitochondrial proteins – Nuclear proteins – Peroxisomal proteins 6  Protein synthesis at the ER example signal sequence: Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala-Thr-Glu-Ala-Glu….  Signal Sequence – SRP interaction Protein synthesis starts with a cytosolic ribosome Signal sequence is first part protein synthesised (N-terminus) Signal sequence recognised by Signal Recognition Particle (SRP) protein complex SRP binds the SRP-receptor on ER membrane Ribosome now bound to ER membrane New protein is guided through translocon (translocation channel) in ER membrane into the ER lumen Signal sequence cleaved via signal peptidase and SRP is recycled 8  Membrane proteins remain in the RER membrane Stop transfer sequences: 20-22 amino acids Stop transfer sequences remain as transmembrane segments of proteins Molecular Biology of Cell: Alberts et al. Fig 12-47  Proteins and RER Tans-membrane proteins remain in ER membrane Other proteins in the lumen of ER Most proteins synthesised in RER are glycosylated – glycoproteins A common preformed 14 sugar oligosaccharide is added to asparagine residue Protein folding takes place Improperly folded proteins are exported from ER and degraded by proteasome in cytosol 10  ER is the starting point for many proteins Reach final destination through a series of intracellular transport steps Transport is via membrane vesicles Proteins are often further processed in vesicles 11  ER – Golgi interactions Protein containing vesicles bud off from ER, migrate and merge with cis-Golgi Further protein maturation occurs in Golgi Movement between cisterna of Golgi via vesicles In trans-Golgi mature proteins packaged in vesicle for specific compartments: sorting office Vesicles transported along specific cytoskeleton pathways 12  Targeting vesicles to specific compartments Trans-Golgi vesicles are coated with specific v-SNARE proteins that allow them to be targeted to vesicle specific compartments target A complementary t-SNARE protein membrane at the intended destination facilities the correct targeting of the proteins  Targeting proteins to lysosome Lysosomal proteins are synthesised in the ER and transported to the trans Golgi network Lysosomal proteins are packaged into vesicles that are targeted to endosomes and phagosomes that converts them into lysosomes Lysosomal proteins are tagged with mannose-6-phosphate in Golgi Mannose-6-phosphate receptor in Mannose 6-phosphate Golgi directs proteins into correct transport vesicles 14 Fate of proteins translated in cytosol 15  Targeting proteins to the mitochondria Targeting happens after translation is completed but before complete protein folding occurs Protein is complexed with chaperone (HSP70) Specific signal sequence recognised by receptor in outer membrane of mitochondria example signal sequence: Met-Leu-Ser-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Ala-Thr-Leu-Cys-Ser-Ser-Arg-Tyr-Leu-Leu…. 16 “postcode” for mitochondria Chaperones take proteins to mitochondrion  Targeting proteins to nucleus Targeting happens after translation and folding is completed in cytoplasmic Nuclear proteins contain Nuclear Localisation Signal (NLS) – Pro-Lys-Lys-Lys-Arg-Lys-Val (PKKKRKV) – basic NLS binds to importin and is transported through the nuclear pore Requires G-protein Ran and hydrolysis of GTP Exportin performs the reverse function to exporting proteins from nucleus 18 Targeting proteins to nucleus - diagram Complex dissociates and nuclear protein is released nucleus nuclear membrane cytoplasm Protein with a NLS  Targeting proteins to peroxisome Peroxin proteins have three amino acid sequence at C-terminus – Ser-Lys-Leu This is recognised by PEX5 and this complex is imported into the peroxisome 20  Summary Specific sequences in proteins allow for them to be targeted to specific compartments Specific proteins bind to each targeting peptide sequences Secreted / membrane / lysosomal proteins have a Signal Sequence – that facilitates the entry into the ER/Golgi system, transport involves the use of vesicles Nuclear / mitochondrial / peroxisome proteins have specific targeting sequences, the targeting occurs after translation is complete in the cytosol 21