Protein Sorting - BCCB2004 Lecture Notes (PDF)

4. Protein Sorting 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 materi...

4. Protein Sorting 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 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 ◼ Post / ZIP codes inform postal services where to send mail ◼ Biology had thought to do this before civilisations had this idea! U.S.A. U.K. MALWMRLLPL LALLALWGPD PAAAFVNQHL CGSHLVEALY LVCGERGFFY TPKTRREAED LQVGQVELGG GPGAGSLQPL ALEGSLQKRG IVEQCCTSIC SLYQLENYCN ◼ Amino acid sequence instructs localisation 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 Post codes on a cellular level ◼ Target sequences – different properties, common mechanism ◼ EXAMPLES ◼ mitochondria ◼ peroxisomes ◼ nucleus (import or export) ◼ endoplasmic reticulum (retention or export) 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 Targeting peroxisomes ◼ C’ terminal ◼ Tripeptide comprising serine (Ser / S), lysine (Lys / K) and leucine (Leu / L) ◼ Uncleaved 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 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 ◼ 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 Mechanisms of protein sorting Translocation into mitochondria ◼ Proteins destined to the mitochondria are maintained in an unfolded state prior to translocation ◼ The translocation channel is to narrow to allow import of folded proteins Created with BioRender.com Translocation into mitochondria Mitochondrial signal sequence Receptor outer membrane Inner membrane precursor protein 1. Binding of precursor to membrane receptor Alberts et al. Translocation into mitochondria Mitochondrial signal sequence 2. Insertion into membrane by the TOM complex TOM protein Receptor outer membrane Inner membrane TIM protein precursor protein 1. Binding of precursor to membrane receptor 3. Translocation into matrix via the TIM complex Signal to enter the matrix is poorly understood TOM – Translocase OUTER membrane TIM – Translocase INNER membrane Alberts et al. 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 4. Complete translocation into matrix requires ATP hydrolysis 3. Translocation into matrix via the TIM complex TOM – Translocase OUTER membrane TIM – Translocase INNER membrane Alberts et al. 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. 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 Translocation into the nucleus Mutation of a single Lys is sufficient to prevent nuclear import Translocation into the nucleus ◼ Folded protein enter nucleus through gated pores ◼ The nuclear pore complex is permeable to molecules ≤ 60 kDa ◼ The larger the protein the slower the rate of diffusion Created with BioRender.com Translocation into the nucleus ◼ Protein ◼ Signal >60 kDa are transported into the nucleus sequence not cleaved ◼ can be anywhere in the protein sequence ◼ also called nuclear localisation sequence (NLS) ◼ Proteins first recognised and bound by protein called nuclear transport protein (importin) Created with BioRender.com Translocation into nucleus ◼ One of the largest & longest lived protein complexes in cells ◼ On average 2000 / cell ◼ Complexity reflects that proteins are both imported and exported through the nuclear pore Translocation into nucleus ◼ Importin / substrate first binds to the cytoplasmic filaments on the nuclear pore complex ◼ FG (Phe-Gly repeats) repeats also act as a diffusion barrier ◼ Importin is able to bind to FG repeats and migrate through the pore ◼ Nuclear basket binds to nuclear cytoskeleton. May also bind to chromatin 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 Translocation in and out of the nucleus ◼ The Ran GTPase provides directionality to nuclear transport Pi Ran GTP Ran.GTP binding to importincargo complex leads to dissociation of the complex GTPase activating protein (GAP) Ran GDP Guanine nucleotide exchange factor (GEF) Ran GTP GDP GTP Ran GDP Ran dependent GTP hydrolysis leads to dissociation of the exportin-cargo-Ran complex 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