Intracellular Organisation, Membrane Traffic

• The key cellular specialisations of eukaryotic cells are: DNA, which is linear (with introns) & bound to proteins; organelles, which includes nucleus & are membrane-bound; reproduction, which occurs by mitosis/meiosis with paired chromosomes; average size is large, which is around 10-100 micrometers.
• • Most eukaryotic cells have the same basic set of membrane-enclosed organelles by the evolution of eukaryotic cells from the combination of ancient archaeon and aerobic bacterium. Because the aerobic bacterium may enclosed into archaeon, from archaeon's expanding membrane and protrusions which enclose bacterium. The protrusion of membranes then eventually fuse with each other to form internal membrane-enclosed compartments, as early organelles.
• • Topology describes spaces that remain unchanged or are similar to each other under any re-formations, such as transfer of a protein without crossing a membrane.
  • Topology is important because it maintains cell environments (chemical balances) that are essential for different mechanisms (e.g. protein transfer).
• • Topological relations of organelles is that cytosol and nucleus share same topology, because nucleus was the cytosol of ancient archaeon so fusing of archaeon encloses nucleus compartment with the same topology as cytosol. Lumen of organelles (i.e. except mitochondria) have the same topology as extracellular space, because it is formed from the protrusions/fusing of ancient archaeon enclosing over an extracellular space.
• • The three key mechanisms for moving proteins between compartments intracellularly are: protein translocation, from cytosol to plastids, mitochondria, endoplasmic reticulum & peroxisomes; gated transportation, from nucleus to cytosol or vice versa; and vesicular transportation, from ER to Golgi apparatus (then to other organelles of cell), peroxisome, late endosome & secretory vesicles.
• • The role of sorting signals is as sorting sequences found on proteins. The role of sorting receptors is to read sorting sequences of proteins & ensure that these proteins are transported to its correct location. This is important because it ensures efficient delivery of proteins for: import into nucleus; export from nucleus; import into mitochondria; import into ER; and return to ER.
• • The key structural specialisations of the ER are: membranous network, which extends from nuclear envelope into much of cytosol; one complex, i.e. enclosed lumen; have rough ER (studded with ribosome) & smooth ER (lacks bound ribosome) lumen regions.
• • The key functional specialisations of ER are: facilitates protein & lipid biosynthesis (i.e. protein folding); and is responsible for intracellular Ca^2+ cell signalling & storage.
• • The mechanisms of translocating soluble proteins into the ER lumen are: post-translational translocation, where peptides are first translated by free ribosomes in cytosol, then is assisted by chaperones to remain unfolded in cytosol before being 'fitted' into protein translocator to be translocated into ER; co-translational translocation, where ribosomes for translating peptides become membrane bound, so peptides are 'fitted' through protein translocator in ER membrane while still being translated.
• • Post-translational translocation occurs by free ribosomes in cytosol synthesising peptide via translation. Peptide chain then binds to chaperones in cytosol, to prevent it from folding. Peptide then binds to protein translocator at ER membrane & activates it. Peptide is then 'fitted' through the protein translocator into ER lumen to be folded into mature protein before being exported out of ER.
• • Co-translational translocation occurs by free ribosome in cytosol starting translation, becoming ER bound due to ER signal sequence on growing peptide which is recognised by SRP (signal recognition peptide). SRP binds to signal, which slows down translation, before binding to SRP receptor on rough ER membrane. Binding activates protein translocator, so growing peptide chain is 'fitted' through translocator into ER lumen. Signal peptidase, closely associated with protein translocator, cleaves off signal sequence after peptide is synthesised, so that it can be folded into mature protein.
• • Similarities between post-translational & co-translational protein translocation is that they both require use of protein translocator for peptides to be in ER lumen.
  • Differences is that co-translational translocation has still-synthesising peptide chains (with ER bound ribosomes), while post-translational translocation has completed peptide chains (with chaperones).
• • The mechanism of translocation of transmembrane proteins into the ER membrane is by co-translational translocation. Occurs by transmembrane peptide exhibiting transmembrane segments recognised by SRP, so SRP binds to SRP receptor that delivers the bound peptide to protein translocator (e.g. Sec61 complex). Lateral gates of protein translocator allows transmembrane segments to move out of translocator laterally into lipid bilayer as the still-synthesising peptide is 'fitted' through translocator. Causing the N- & C-terminus regions of non-transmembrane segments to emerge in cytosol or ER.
• • The orientation of transmembrane protein is determined at the time of translocation into the ER membrane by which terminus (N or C) having the most flanking positively charged amino acid. Also by whether N-terminal peptide length is long & folded. Because more positively charged amino acid terminus emerges on cytosolic side of ER membrane.
• • Most proteins synthesised in rough ER are glycosylated by still-synthesising peptides having oligosaccharide precursor transferred to Asn residue for N-glycosylation at amine group. This is catalysed by enzyme oligosaccharyl transferase binding to protein translocator. This forms mature protein with N-linked oligosaccharide in ER lumen.
  • Proteins are glycosylated because this helps ensure protein regulation when folding occurs in ER, since this acts as identifiers of protein quality control.
• • Protein folding is regulated in the ER lumen by synthesised peptide with oligosaccharide precursor undergoing two glucose trimming by glucosidase enzyme after glycosylation. Glycosylated peptides with one terminal glucose is recognised by chaperone calnexin at ER membrane, so calnexin binds to the terminal glucose. Glucose then trimmed by glucosidase again to allow peptide to fold. Glucosyl transferase enzyme then binds to folded protein and checks if protein folding is correct.
• • Protein folding is regulated in the ER because if folding is correct, this allows mature proteins to be exported from ER. However, if folding is incorrect, glucosyl transferase will add another terminal glucose onto the N-linked oligosaccharide of peptide, so that it can be recognised & bound to calnexin again. So that the proteins can have another chance to correctly fold. Because if the misfolding keeps happening after several cycles, peptides are targeted for degradation in the proteasome.
• • Mitochondrial protein-precursor synthesis occurs in the cytosol, because cytosol has free ribosomes for protein translation. Mitochondrial protein-folding occurs in mitochondrial matrix, because folded proteins in mitochondria are used locally in the organelle.
• • Protein translocation into the mitochondrial matrix occurs by completed peptide chains, with chaperones, binding to TOM complex receptor on outer membrane, because TOM recognises N-terminal signal sequence on peptide. TOM-bound peptide then translocates across outer membrane until TOM lines up with TIM complex on inner membrane. Peptide translocate across the TOM-TIM lined up channel into mitochondrial matrix, using ATP hydrolysis, membrane potential & redox potential to provide energy. Signal peptidase in matrix then cleaves off signal sequence of peptide, to allow protein folding.
• • The role of signal sequence in protein translocation into mitochondrial matrix is to be recognised by & bind to TOM to initiate translocation, because this allows the protein translocators to line up for peptide to be 'fitted' through & translocate into the matrix to be folded for mitochondrial functions.
• • The TIM & TOM need to line up to allow entry of peptide from cytosol into mitochondrial matrix, because mitochondria have double membrane from origin of endosymbiont aerobic bacterium.
• • Nuclear protein synthesis and folding occurs in cytosol, due to free ribosomes available in cytosol.
• • The mechanism for molecule transportation between cytosol and interior of nucleus is gated transportation. This occurs by differential localisation of Ran GDP (in cytosol) & Ran GTP (in nucleus), driving directionality to nucleus import and export. This is because Ran GAP (GTPase Activating Protein) is abundant in cytosol, so hydrolyses GTP to GDP, allowing GDP to travel down concentration gradient into nucleus. Similarly, Ran GEF (Guanine exchange factor) is bound to chromatin in nucleus so is abundant & exchanges GDP with GTP, allowing GTP to travel down concentration gradient into cytosol.
• • The basic structure of nuclear pore consists of: scaffold nucleoporin, first layer of transmembrane protein that forms a ring around nuclear pore; channel nucleoporin, second layer of transmembrane protein that forms a ring around nuclear pore & forms disordered regions in nuclear pore; cytosolic fibrils, long extensions from scaffold nucleoporin on cytosolic side & forms nuclear basket from scaffold nucleoporin on nucleus side.
• • Nuclear import is regulated by GTPase switch. Cargo with nuclear localisation signal, for import, binds to nuclear import receptors in cytosol. The cytosolic fibrils recognise receptor-cargo complex & allows cargo to be transported into the nucleus through nuclear pore. Ran-GEF exchanges Ran GTP with cargo, causes receptor to release cargo in nucleus & bind to GTP. Ran-GTP-receptor complex get transported out of nucleus via nuclear pore as Ran-GTP goes down its concentration gradient. Ran-GAP hydrolyses GTP to GDP, so receptor releases GTP & is available to transport more cargo.
• • Nuclear export is also regulated by GTPase switch. An empty nuclear export receptor is recognised by cytosolic fibrils & is transported into nucleus via nuclear pore. Ran-GEF activates Ran-GTP binding to receptor, while cargo with nuclear export signal also binds to receptor at different site to Ran-GTP. Cargo-GTP-receptor complex get exported out from nuclear pore, by Ran-GTP going down concentration gradient. Ran-GAP hydrolyses Ran-GTP to Ran-GDP, causing receptor to release Ran-GDP and cargo in cytosol at the same time. Receptor can then be transported back for more cargo in nucleus.
• • Proteins can move between organelles by vesicle budding and fusion by the topology of vesicles & target organelle being the same, because this allows material/cargo to be exchanged but organelle environment to be the same since no membranes were crossed.
• The three pathways of vesicular transport are: secretory pathway, forward pathway; endocytic pathway, from plasma membrane; and retrieval pathway, reverse pathway.
• • The organisation of inner & outer leaflets of membrane lipid bilayer is maintained during vesicle budding by the cytosolic leaflet, lumen leaflet & soluble/membrane material of donor organelle being maintained in vesicle in same position. The inner & outer leaflets are also maintained during fusion by coating assembly allowing bud formation, leading to vesicle formation, leading to uncoating of vesicles for fusion.
• • The similar key features of secretory & endocytic pathways is that both transport cargo by vesicles. The different key features is that secretory pathway allows anything made in the cell to be transported to another location in or out of the cell. While endocytic pathway only transports molecules that will be permanently taken up by the cell.
• • The various types of vesicle coat proteins include: clathrin, for vesicles transporting from plasma membrane (i.e. endocytosis), Golgi & endosome; COPI, for vesicles in retrieval pathway (i.e. from Golgi to ER); COPII, for vesicles in secretory pathway (i.e. from ER to Golgi); and retromer, for vesicles transporting from endosomes to Golgi.
• • The different types of vesicle coat proteins are important because they give specificity to vesicular transport & facilitates efficient transport between different compartments, through a lock & key mechanism with adaptor proteins. Further modifications in building the subunits of coat proteins gives rise to higher specificity.
• • Specific cargo molecules are selected during vesicle budding by cargo molecules binding to cargo receptors. Because the binding of this cargo to receptor recruits adaptor proteins to cargo-receptor complex, allowing vesicle coat proteins to bind to adaptor proteins through lock & key mechanism, ultimately selecting the cargo receptors with cargo molecules for transport.
• • The mechanism for the formation and budding of clathrin vesicles occur by a cargo first binding to cargo receptor on the membrane of donor organelle. This complex then recruits adaptor proteins, which then recruit & bind to clathrin through lock & key mechanism, enabling a slight curvature to form on the membrane's surface. Further bud formation leads to vesicle formation with only a small nick connecting the vesicle & donor organelle. Membrane-bending & fission proteins then strips this nick & pinches off the vesicle. Clathrin & adaptor protein shed from vesicle, for naked transport vesicle.
• • Slight buds form on membrane surface when clathrin is recruited because triskelion shape of clathrin causes the curvature, as well as additional membrane bending force being generated when clathrin binds.
• • The molecular basis for specific targeting of vesicles to specific compartments is the interaction of Rab molecules on vesicles & target membrane, because Rab molecules ensure specificity of vesicle & target membrane for correct cargo deposit.
  • The molecular basis for specific fusion of vesicles to specific compartments is the interaction of vSNARE (vesicle SNARE) & tSNARE (target SNARE) proteins, because SNAREs pair up to dock vesicles on target membrane & catalyses the fusion of lipid bilayers of both the vesicle & target membrane.
• • SNAREs fuse vesicles and target membranes by the pairing of vSNARE & tSNARE forcing bilayers to be closed & expels water from the interface between the bilayers. The two interacting cytosolic leaflets then forms connecting stalk by its lipid molecules flowing between the two membranes. The lipids of the two non-cytosolic leaflets make contact with each other to form new lipid bilayer, which widens fusion zone by flattening the budding & SNAREs moving laterally. This new bilayer from non-cytosolic leaflets is then ruptured, completing fusion process.
• • Proteins leave the ER through secretory pathway by inactive Sar1-GDP coming to ER membrane & Sar1-GEF on ER membrane exchanges GDP for GTP, activating Sar1-GTP, triggering conformational change of exposing amphiphilic helix in Sar1. Helix is then inserted into cytosolic leaflet, initiating membrane bending, while Sar1-GTP recruits adaptor proteins Sec23&24 causing ER membrane to start curvature as Sec24 recognises cargo. Sec23&24 forms inner COPII coat & recruits Sec13&31 to form outer COPII coat. Sec13 & 31 assembles into symmetrical cage to enclose COPII-coated vesicle that is pinched off.
• • Proteins enter the cis-Golgi through secretory vesicle from ER, it then moves through cis-, medial-, trans-cisterna in Golgi compartments to get sorted and exit trans-Golgi in a vesicle to be delivered to appropriate locations (i.e. lysosome, plasma membrane, secretory vesicle).
• • Similarities between constitutive & regulated secretory pathway are that both deliver contents via vesicles to plasma membrane. Both pathways are also initiated after sorting at trans-Golgi.