Cell Biology: Protein Sorting

Cell Biology: Protein Sorting

1. Post-translational Modifications

   • Glycosylation
     • Adding and processing of oligossacharide chains
     • 90% N-linked to amide of asparagine in ER lumen, and 10% O-linked to hydroxyl of serine in Golgi
     • Mechanism for N-linked oligosaccharides
       • Oligossacharide precursor is synthesised on a dolichol carrier, forming a complex attached to the ER membrane that faces the lumen.
         • - Oligosaccharide precursor is composed of 14 residues (3 glucose residues, 9 mannose residues and 2 N-acetylglucosamine (GlcNAC) residues -> Glc3Man9(GlcNAC)2) - Product of the complex set of reactions is a dolichol pyrophosphoryl oligosaccharide - Dolichol is a long-chain polyisoprenoid (isoprene is a branched-chain unsaturated hydrocarbon; isoprenoid is the plural) lipid that is firmly embedded in the ER membrane
         • Entire oligosaccharide precursor is then transferred to an asparagine residue on a nascent polypeptide as it emerges into the ER lumen
           • - Only asparagine residues in the tripeptide sequences Asn-X-Ser and Asn-X-Thr (where X is any amino acid except proline) are substrates for oligosaccharyl transferase, not all of these sequences are glycosylated, however. - Three glucose residues, last to be synthesised, act as signal that oligosaccharide synthesis is complete and is ready to be transferred onto a protein
           • Carbohydrate structure is modified in the ER and Golgi of Secretory Pathway
             • - The 5 conserved residues consists of 3 mannose and 2 N-acetylglucosamine (GlcNAC) residues
     • Functions
       • Mediates proper folding in some proteins
       • Cell-to-cell recognition and adhesion (when attached to plasma membrane proteins)
       • Stabilises some secreted glycoproteins (w.r.t. temperature, pH, etc.)
       • Enzyme function
         • In terms of substrate affinity and enzyme kinetics
       • Targeting signal
         • eg. M6P to lysosomal enzyme for recognition by M6P receptor in TGN, plasma membrane and endosome.
   • Disulfide bonds
     • For eukaryotes, formed AND rearranged only in the lumen of RER
       • Hence proteins with disulfide bonds are either secretory proteins or formed at exoplasmic domains of membrane proteins
         • - Exoplasmic: side of membrane facing away from cytosol
         • Latter is because most cellular compartments (including cytosol) are reducing environments, hence disulfide bonds would be unstable
       • Proteins translated by free ribosomes lack disulfide bonds, and are stabilised by other interactions
       • Rearrangement is due to disulfide bonds formed in sequential order as cysteine residues are added, which may not be most stable conformation
         •  Disulfide bonds generally form in a specific order, first stabilizing small domains of a polypeptide, then stabilizing the interactions of more distant segment  
         • Rearrangment also catalysed by PDI, through similar thiol-disulfide transfer reactions
     • Functions as intra- and inter-molecular bond to stabilise protein
     • Formed by oxidation of two thiol groups from cysteine residues
     • Mechanism
       • Ionised (S-) form of cysteine thiol in substrate protein reacts with disulfide bond in PDI, forming a disulfide-bonded PDI-substrate intermediate
         • Second ionied cysteine then reacts with this bond, forming a disulfide bond within the substrate protein and releasing reduced PDI
           • Oxidised PDI regenerated via luminal protein Ero1
     • Catalysed by
       • Protein Disulfide Isomerase (PDI)
   • Complete folding of polypeptide chains and assembly of multimeric proteins in ER
   • Specific proteolytic cleavages in ER, Golgi and secreory vesicles

2. Organelles

   • Endoplasmic Reticulum (ER)
     • Protein Translocation
       • Cotranslational Translocation
         • Simultaneous translocation of protein into ER lumen and synthesis of the protein
         • Components
           • Signal Recognition Particle (SRP)
             • Ribonucleoprotein (combination of protein and RNA)
             • Binds to ribosome and nascent polypeptide during translation through recognition of the signal sequence, which induces a conformational change around the Hinge of the SRP and activates its Translational Pause Domain to cause translation arrest
           • N-terminal signal sequence
           • SRP receptor
             • Integral membrane complex of α and β subunits. Interaction of SRP receptor with SRP mediates insertion of nascent protein into Sec61 translocon
           • Sec61 translocation channel / translocon (for eukaryotes)
             • Protein complex made of α, β, γ subunits
           • Signal peptidase
             • Cleaves signal sequence from nascent protein during/after entry into ER lumen
         • Mechanism
           • Specific process due to specificity of SRP to signal sequence and of SRP receptor to SRP
           • Translation begins in cytosol by a free ribosome
             • SRP binds to ER signal sequence once it emerges from ribosome
               • SRP delivers ribosome-polypeptide complex to SRP receptor in ER membrane. Binding of GTP to SRP and SRP receptor strengthens this interaction
                 • Sec61 translocon "opens" as ribosome-polypeptide complex is transferred to it and the signal sequence (5' N-terminal) is inserted first. This transference releases the SRP and SRP receptor and the bound GTP is hydrolysed, allowing them to initiate another translocation
                   • Release of SRP resumes translation, and the elongating polypeptide is pushed by the ribosome bound to the translocon. Signal sequence may then be cleaved depending on the type of protein synthesised (see note)
                     • For Type I transmembrane proteins, ER signal sequence is cleaved by signal peptidase in ER lumen and is rapidly degraded. For Type II transmembrane proteins and sone polytopic proteins the signal sequence is not cleaved and hence they are called signal anchor sequences More at https://en.wikipedia.org/wiki/Protein_targeting
                     • Within the ER, the protein is first covered by a chaperone protein to protect it from the high concentration of other proteins in the ER, giving it time to fold correctly
                       • Translation completes, ribosome is released, translocon closes and resumes its native folded conformation
       • Post-Translational Translocation
         • Translocation of proteins into the ER lumen after the protein has been completely synthesised.
         • Disadvantage: Secondary structure of protein sometimes needs to be unfolded in order to bind to transporters -> Inefficient
         • Common to bacteria, yeast and small secreted proteins (<100 amino acids) in mamallian cells
         • Mechanism
           • Signal sequence interacts directly with translocon
             • Polypeptide enters the translocon, initially slides freely in and out
               • BiP chaperones (localised to lumen) invoke ATP hydrolysis to provide energy to racheting mechanism that pulls polypeptide into ER lumen
                 • Polypeptide chain is pulled into ER lumen via a ratcheting mechanism that requires ATP hydrolysis by chaperone BiP
       • GPI-anchored Proteins
         • Allows for diffusion across phospholipid bilayer
           • Otherwise protein would be immobilised by membrane-spanning alpha helices with C-terminals in the cytosol interact with the cytoskeleton
         • Mechanism
           • Precursor protein is synthesised like a Type I single-pass transmembrane protein (stop-transfer/membrane-anchor sequence and a cleaved N-terminal signal sequence)
             • Specific transamidase cleaves the precursor protein near the stop-transfer sequence within the exoplasmic facing domain, and transfers the carboxyl group of the C-terminus to the terminal group of a preformed GPI anchor
               • Mature GPI-anchored protein produced, it is further processed through the secretory pathway (vesicles and Golgi apparatus) and eventually remains attached to the extracellular region of the plasma membrane
     • Function
       • Ca2+ Storage
       • Synthesis, folding and maturation of most secreted and transmembrane proteins eg. plasma, nuclear membrane proteins
     • Homogenised to produce
       • Apply pressure and mix to break ER apart, forming small lipid vesicles, some with ribosomes attached and some without.
       • Microsomes
         • Rough microsomes can be used in cell free protein translation and translocation assay
         • Centrifugation with sucrose solution of increasing concentration separates rough and smooth microsomes due to sedimentation coefficient
   • Mitochondria
     • Protein Import
       • Proteins are sometimes unfolded by chaperones before entry into mitochondria / chloroplasts
       • Mechanism for matrix protein
         • Precursor proteins are maintained in unfolded/partially unfolded state by cyotosolic chaperones such as Hsc70
           • Matrix targeting sequence guides protein to an import receptor near a Contact Site, and the protein is transferred into a general import pore (Tom40)
             • Translocation occurs through this channel and adjacent channel in inner membrane
               • Binding by matrix chaperone Hsc70 and its ATP hydrolysis drives import into the matrix.
                 • Targeting sequence is cleaved by matrix protease, causing release of Hsc70
                   • Protein folds into mature conformation, some require help from matrix chaperonins
     • Proteins
       • Nuclear-encoded
         • Synthesised in the cytosol
           • Mt has receptor proteins on both membranes that recognise specific targeting sequences at N-terminal of proteins
             • Active process
         • Different pathways needed for
           • Matrix (2/3 of total)
             • Has amphipathic targeting sequences in α-helical conformation
           • Others
             • Inner membrane
               • 3 methods
             • Outer membrane
             • Intermembrane space proteins
               • 2 methods
             • Generally require stop transfer sequence / more targeting sequences
       • Mitochondrial
         • OXPHOS proteins
           • Occurs at electron transport chain
           • eg. NADH dehydrogenase, cytochrome oxidase and ATP synthase
         • Synthesised in the matrix
   • Peroxisome
     • Protein Import
       • Peroxisomal Targeting Signals (PTS)
         • PTS1 (Major, C-terminal)
           • Only example I have of C-terminal targeting sequence so far
           • Targets for most peroxisomal matrix proteins
           • Most common is serine-lysine-leucine (SKL)
         • PTS2 (Minor, N-terminal)
       • Peroxisomal proteins, however, can be imported in their mature conformations
       • Mechanism
         • Similar to co-translational translocation, with receptor Pex5 binding to PTS1, bringing it to receptor Pex14 on peroxisomal membrane, then transferring to membrane translocon (unknown)
     • Zellweger Syndrome
       • Failure to import proteins into peroxisome due to either membrane receptor (Pex2) or cytosolic receptor (Pex5) mutation, results in accumulation of long fatty acid chains in cells and "empty" peroxisomes
     • Proteins
       • All nuclear-encoded, synthesised in cytosol
       • Enzymes that catalyse oxidative reactions of fatty and amino acids
         • Produces H2O2 as by-product, converted to H2O and O2 by catalase
     • Enlarges by addition of proteins, divides to form new ones

3. Important terms

   • Signal (targeting) Sequence
     • Mostly at N-terminus, except for peroxsiomal proteins (most C-terminus)
     • Signal Peptidase
       • Cleaves signal peptides during transport into target organelles
       • NOT for NLS entry into nucleus

4. Quality Control

   • Example
     • Rhodopsin Class II mutation
       • Rhodopsin is the primary pigment found in photoreceptor cells
       • Class II mutant opsins are misfolded (probably due to ineffective glycosylation) and is retained in the ER, whereas WT interacts with chaperones to be expressed on the plasma membrane
         • Accumulation of misfolded opsin causes ER stress and induces UPR, which if does not alleviate ER stress results in cell apoptosis
         • Usage of 9-cis-retinal (new) instead of 11-cis-retinal (actual) chaperone allowed mutant to fold properly to be expressed on plasma membrane
   • Unfolded Protein Response (UPR)
     • Causes
       • Disturbance of Ca2+ homeostasis
       • Imbalance of demand and capacity of protein-folding apparatus
     • Reduces accumulation of unfolded proteins by
       • Activation of ER-localised chaperones
       • Slowing of protein synthesis
       • Enhance protein degradation

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