Protein Sorting (Part II) BCCB2004 PDF

5. Protein Sorting (part II) BCCB2004 – Foundations of Cell Biology Dr. Carl Mousley [email protected] 305.117 Ext 5617 WARNING This material has been reproduced and communicated to you by or on behalf of Curtin University in accordance with section 113P of the Copyright Act 1968 (the Act)...

5. Protein Sorting (part II) BCCB2004 – Foundations of Cell Biology Dr. Carl Mousley [email protected] 305.117 Ext 5617 WARNING This material has been reproduced and communicated to you by or on behalf of Curtin University in accordance with section 113P of the Copyright Act 1968 (the Act) The material in this communication may be subject to copyright under the Act. Any further reproduction or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice. Outline ◼ Understand the characteristics of post codes that target proteins to organelles ◼ Understand ◼ Into how proteins are transported: mitochondria ◼ Into peroxisomes ◼ In and out of the nucleus ◼ Targeting the endoplasmic reticulum Protein sorting Some proteins are synthesised in the cytoplasm but function in other cellular compartments HOW? Created with BioRender.com Protein sorting ◼ For a cell to function properly each protein must be directed to the correct cellular membrane or compartment ◼ e.g. mitochondria, lysosome, endoplasmic reticulum, cytosol, nucleus, plasma membrane ◼ The process of directing each protein to a particular destination is protein sorting or targeting. ◼ It is essential to a cells function, organisation, and survival (as critical as DNA replication/ transcription & translation) Protein sorting ◼ How does a cell know how to target a protein to the appropriate organelle??? Created with BioRender.com Post codes on a cellular level ◼ A polypeptide contains the information to direct it to the appropriate organelle ◼ Protein ◼ Target post codes – targeting peptides peptides ◼ Pre-sequences ◼ Internal targeting peptides Internal targeting peptide Pre-sequences N-terminal - mostly!!! C-terminal continual patch Targeting the mitochondria Pre-sequence Nterm - M R S L R Q S I R F F K P A T R T L C S S L Y L L ◼ N’ terminal ◼ +ve charged amphipathic helix Basic amino acids (Arg/ Lys) are on the same face of the helix ◼ Amphipathic = hydrophilic and hydrophobic properties ◼ Cleaved Translocation into mitochondria Mitochondrial signal sequence TOM complex 2. Insertion into membrane by the TOM complex Receptor outer membrane Inner membrane TIM complex precursor protein 1. Binding of precursor to membrane receptor ATP → ADP + Pi 3. Translocation into matrix via the TIM complex 4. Complete translocation into matrix requires ATP hydrolysis 5. Cleavage of signal sequence by signal peptidase TOM – Translocase OUTER membrane TIM – Translocase INNER membrane Alberts et al. Targeting peroxisomes ◼ C’ terminal ◼ Tripeptide comprising serine (Ser / S), lysine (Lys / K) and leucine (Leu / L) ◼ Uncleaved Created with BioRender.com Translocation into peroxisomes ◼ PEX-receptor binds to the PTS ◼ Receptor-cargo complex is targeted to the peroxisome membrane ◼ PEX-receptor is incorporated into the PEX translocon ◼ Cargo is translocated ◼ PEX-receptor dissociates from cargo Created with BioRender.com Targeting the nucleus ◼ Signal sequence ◼ Monopartite ◼ Bipartite ◼ Signal..... P P K K K R K V……….. K R -[PAATQQAGQA]- K K K K Patch A basic patch is formed in the folded protein Translocation into nucleus ◼ Nuclear transport receptor (importin) recognises NLS – cargo complex associates with cytoplasmic filaments ◼ Receptor – cargo complex associates with and walks along FG repeats ◼ Receptor ◼ Receptor-cargo complex migrates through the pore – cargo complex dissociates in the nucleus ◼ Receptor Created with BioRender.com Translocation out of the nucleus The same as import but in reverse!!! ◼ Many proteins shuttle between the cytosol and nucleus ◼ Proteins possess nuclear export signals: LxxxLxxLxL (leucine rich most common) ◼ Nuclear export receptor (exportin) recognises NES – cargo complex associates with nuclear pore complex ◼ Exportin ◼ Receptor-cargo complex migrates through the pore – cargo complex dissociates in the nucleus Created with BioRender.com ◼ Receptor Targeting the endoplasmic reticulum The endoplasmic reticulum is the entry point to the secretory and endocytic pathway Created with BioRender.com Targeting the endoplasmic reticulum; the entry point to the secretory pathway Approx. 1/3 of proteome is secreted (up to 2/3 in a professional secretory cell) Endoplasmic reticulum Golgi apparatus Endosomes Lysosomes Plasma membrane Targeting the endoplasmic reticulum ◼ Pre-sequence 13 – 36 amino acids N-region H-region C-region hydrophobic core K / R before H-region Enriched in hydrophobic (F/I/L/V/W/Y) amino acids contains polar residues, marks the cleavage site of cleavable sequence Targeting the endoplasmic reticulum ◼ Examples Human Preproinsulin MALWMRLPLLALLALWGPDPAAAFV… Bovine Growth Hormone MMAAGPRTSLLLAFALLCLPWTQVVGAF… Bee Promellitin MKFLVNVALVFMVVYISYIYAAP… Targeting the endoplasmic reticulum ◼ The ◼ It signal peptide may or may not be cleaved will become the first transmembrane domain if it is not cleaved Created with BioRender.com Targeting the endoplasmic reticulum ◼ Three components required to translocate protein with ER signal sequence into the ER ◼ Signal recognition particle (SRP) ◼ Signal recognition particle receptor (SRP receptor) ◼ Translocation ◼ Also channel called a translocon Targeting the endoplasmic reticulum ◼ Signal recognition particle ◼a protein/RNA complex that recognises the amino terminal signal sequence on protein ◼ binds ◼ uses ◼ SRP to SRP receptor on membrane of endoplasmic reticulum GTP and plays major role in targeting protein to endoplasmic reticulum receptor ◼ Comprise ◼ The α and β subunits α subunit binds GTP and SRP Targeting the endoplasmic reticulum SRP = 7S RNA and six proteins ‘ribonucleoprotein’ Alu domain pauses peptide elongation on ribosome S domain Binds signal sequence and signal recognition particle receptor Halic & Beckmann (2005) Targeting the endoplasmic reticulum Yellow = 40S ribosome Blue = 60S ribosome Red = SRP SRP associated with the ribosome Halic & Beckmann (2005) Targeting the endoplasmic reticulum Targeting the endoplasmic reticulum Targeting the endoplasmic reticulum Targeting the endoplasmic reticulum Structure of the ribosome bound to the ER translocon Targeting the endoplasmic reticulum The ‘rough endoplasmic reticulum’ represents ribosomes bound to the ER translocon Integral membrane proteins are incorporated into the membrane co-translationally When a TMD is inserted into the translocon it can move laterally into the ER membrane Integral membrane proteins are incorporated into the membrane co-translationally TMD TMD The translocon opens laterally to the ER membrane TMD partitions into the ER membrane TMD Integral membrane proteins are incorporated into the membrane co-translationally Proteins are N-glycosylated as they are translocated into the ER Created with BioRender.com Disulphide bonding - Disulphide bonding takes place in the endoplasmic reticulum - It is catalysed by protein disulphide isomerase - Disulphide bonds increase protein stability Created with BioRender.com Summary ◼ Describe the characteristics of post codes that target proteins to organelles ◼ Describe ◼ Into how proteins are transported: mitochondria ◼ Into peroxisomes ◼ In and out of the nucleus ◼ Into the ER