Chapter 15Intracellular compartments and transportThis material is critical to understanding • a wide variety of lysosomal storage diseases• a wide variety of protein sorting diseases• atherosclerosis/ hypercholesterolemia• secretion of key proteins such as insulin and immunoglobulins• biotechnology involving secreted proteins• potential new therapeutics for Alzheimer’s and prion diseasesObjective: Understand protein sorting in a cell,exocytosis, and endocytosisPart%1%Be%able%to:• Explain the difference between necessary and sufficient• Interpret data and determine if something is necessary, sufficient, or both• Describe the nuclear pore• Diagram the Ran GTP/GDP cycle• Describe the mechanism that inserts proteins in the endoplasmic reticulum, for both soluble and transmembrane proteins, naming the key components• Diagram clathrin-mediated formation of a vesicle from amembrane• Diagram SNARE-mediated fusion of a vesicle with amembrane• Explain the difference between endocytosis and exocytosis• Draw the chemical structure of a disulfide bond in areduced and oxidized stateWhat is the fate of each protein after synthesis?Most proteins function in definedlocations within a eukaryotic cellCompartmentalizationWhat are these compartmentsand how do newly synthesizedproteins get there?Cellularcompartmentalizationis achieved by manydifferent membrane-enclosed organellesSelectively-permeablemembranesPrimary functions of the membrane-enclosedcompartments of a eukaryotic cellCompartmentCytosolNucleusERGolgiLysosomesEndosomesMitochondriaChloroplastsPeroxisomesMain FunctionMetabolic pathways, protein synthesisContains main genome, DNA/RNA synthesisSynthesis of most lipids, protein distributionProtein and lipid modification for distributionIntracellular degradationSorting of endocytosed materialsATP synthesis - oxidative phosphorylationPhotosynthesisOxidation of toxic compounds See text and/or glossary for reviewProtein sorting:Energy-dependentmechanisms forprotein entry intoan organelle fromthe cytosolSignal sequences are necessary and sufficient for protein sortingFor some signal sequences, structural features are moreimportant than specific amino acid sequenceSignal sequences direct proteins to the correct organelleTransport through nuclear poresThe nucleus has twomembranesThe outer membrane iscontinuous with the ERNuclear pores are“gates” through bothmembranes of thenucleus that allowmovement ofmolecules in bothdirections:15_08_nuclear_pore.jpgA nuclear pore is a complex of ~30 different proteins;total of ~450 protein moleculesSmall molecules can freely diffuse through the nuclear porebut large molecules like proteins cannot pass without anappropriate sorting signal - nuclear localization signal(short stretch of lysines/arginines, positive charges) andhelper proteins (nuclear transport receptors)15_09_pore_transport.jpgProtein transport through nuclear pores is activeGTP hydrolysisProteins remain in their fully foldedconformation throughout the transportprocessTransport across mitochondrial and chloroplast membranesProteins must be unfolded during transportChaperonins help re-fold the protein after transportN-terminal signal sequence (structure/charge)15_11_ER.jpgER is the most extensive membrane system in the celland is also the entry point for proteins destined for severalother organelles and the cell exteriorTransport across ER membraneTwo kinds of proteins are transferred from cytosol to ER1. Soluble proteins• Transferred completely to the ER lumen• Destined for secretion out of the cell or to the lumenof another organelle (not mitochondrion or plastid)2. Transmembrane proteins• Partly translocated into ER, embedded in membrane• Destined to reside in ER or the membrane ofanother organelle or plasma membraneBoth types are initially directed to the ER by an ER signalsequence (N-terminal stretch of hydrophobic amino acids)Retention in ER requires a signal sequence at C-terminus(KDEL)Insertion into the ER begins before translation is completedSignal recognition particles (SRPs) in the cytosol and SRPreceptors in the ER membrane provide specificityThe signal peptide remains bound to thetranslocation channel while the rest of theprotein is threaded through as a loop. Oncecompleted, the signal peptide is cleaved.Transport of a soluble protein into the lumen of the ERIntegration of a transmembrane protein into the ERmembraneOrientation will be maintained in subsequent vesicle budding/fusionIntegration of a multi-pass transmembrane proteinIn this example, an internalstart-transfer sequence is usedand the protein is not cleavedTransmembrane protein topologiesComplex topologies can be achieved through variationsand combinations of the basic mechanisms outlined inthe previous two slides. Several of the membranetransport proteins we have discussed earlier thissemester have as many as 12 membrane-spanningdomains.For additional review and practice see Question 15-4Transport by vesicles Endomembrane systemVesicular transport - How is specificity achieved? Specificity is required for the cargo and the destinationEach transport vesicle that buds from a given membranemust take only those proteins appropriate for its destinationand fuse only with the appropriate membraneRecognition events depend on proteins associated with themembranes of each type of vesicle.15_18_Clathrin_EM.jpgSpecialized proteins coat the cytosolicsurface of vesicles: coated vesiclesThe protein coat shapes themembrane into a bud and helpscapture moleculesClathrin-coated vesiclesClathrin-coated pitVesicular transport - specificity for cargoTransport receptors recognize specific cargo proteinsAdaptins trap transport receptors and anchorthem to clathrin.Plasma membrane-specific adaptinsGolgi-specific adaptinsVesicular transport - specificity for the target membraneA vesicle loaded with specific cargo proteins must recognizeand fuse only with the appropriate target membraneSurface molecular markers are incorporated into vesiclemembranes and these are recognized by complementaryreceptors on the target membrane. The markers andreceptors are a family of related transmembrane proteinscalled SNARESVesicle markers: v-SNARESTarget receptors: t-SNARESSNARESNAP ReceptorSNAP=Soluble NSF Attachment ProteinNSF=N-ethylmaleimide Sensitive FactorThis historical derivation is for amusement only!15_20_SNAREs.jpgVesicular transport - specificity for the target membraneVesicle fusion requires additional factors and