Macromolecular Transport LDL Transferrin and Ig RME I 3 Major Processes for Internalization of Macromolecules a Endocytosis i Receptor mediated Endocytosis RME ii Potocytosis b Phagocytosis II Receptor mediated Endocytosis a Over 50 different molecules imported by RME b Common uptake mechanism i Clathrin coated vesicles III LDL Transport a LDL uptake into cells b 1985 Nobel Prize Brown and Goldstein c Specificity for uptake resides in specificity of receptor binding activity i Familial hypercholesterolemia human genetic disease causes too much circulating LDL 1 Hyper too much and emia disease ii Defective RME into cells fails to clear LDL from blood c Low Density Lipoprotein LDL particles i Core 1500 cholesterol esters ii Phospholipid monolayer unesterified cholesterols iii Apoplipoprotein B receptor ligand IV RME of LDL to in to a RME of LDL Experiment i LDL particle coupled ferritin label visible EM ii LDL particles bind to cell surface receptors iii Cluster over coated regions of membranes Internalized in vesicles iv b LDL Apo B in blood binds LDL receptor LDLR i LDL receptor 1 7 finger ligand binding site stabilized by Cys Cys disulfide bonds 2 EGF precursor like B propeller domain important for pH dependence of LDL binding 3 O linked glycosylations maintain receptor erect 4 Single alpha helical transmembrane domain anchor 5 Cytoplasmic domain important for RME NPxY signal c LDL receptors w bound LDL cluster over clathrin coated regions of membrane i AP2 assembly particles link receptor cytoplasmic domain NPxY motif to clathrin coat d Clathrin coated pits invaginate i Pull in region of membrane with LDLR complex e Clathrin coated pits pinch off PM as clathrin coated vesicles i Clathrin coated vesicles 1 Basket formed of hexagonal structures with some pentagons 1 Assembly particles bind globular head of clathrin heavy chains 2 AP mediate interaction geodesic dome 2 Clathrin protein triskelion 3 heavy chains and 3 light chains 3 Arms associate to form lattice 4 Flat lattice invaginates to form basket coat around membrane enclosed vesicle ii Vesicle formation b w clathrin coat and cytoplasmic domain of receptor AP2 binding motifs YXX LL or NPxY motifs 3 Dynamin associated with the pit neck pinches off the vesicle a Dynamin GTPase similar to ATPase b Wraps around and forms neck on pit c GTPase activity associated with pinching off vesicle f Coated vesicle becomes uncoated i Clathrin dissociated to triskelions and recycled to PM ii Uncoated vesicle called primary endosome or early endosome iii Primary endosome fuses with sorting vesicle to form late endosome g H ATPase V class pumps in late endosome lower pH to 5 i Low pH causes LDLR conformational change releases LDL h Late endosome sorting mechanism separates LDL from receptor i Receptors recycled to PM 1 Recovered in vesicle that pinches off endosome and delivers receptors to PM ii LDL remains in late endosome vesicle i Late endosome fuses with lysosome i LDL degraded by lysosomal enzymes ii LDL degradative products recovered as nutrients by transport into cytoplasm iii Digestion of contents by lysosomal enzymes 1 Apo B amino acids 2 Cholesterol esters cholesterol PM bile acids steroids 3 Phospholipids fatty acids H H j GFP Green Fluorescent Protein i 2008 Nobel Prize in Chemistry to Shimomura Chalfie and Tsien ii Naturally fluorescent jelly fish protein 25 kDa iii Can be fused to target protein through cloning to make chimeric protein iv When expressed in cell GFP target protein emits visible light v GFP clathrin reveals activity of coated pit coated vesicle formation V Familial Hypercholesterolemia a 4 Classes of LDLR Mutations i Synthesis ii Transport from ER to Golgi iii Binding of LDL i Receptors fail to cluster efficiently over coated pits because receptor iv Clustering in coated pits b LDLR cytoplasmic domain mutations tail does not interact with AP2 ii One mutation disrupts NPxY motif c Statin fiber treatment promotes LDL uptake i Statin drubs e g Lipitor inhibit cell s ability to synthesize cholesterol 1 Inhibit HMG CoA reductase in cell s pathway for making cholesterol 2 Only source of cholesterol LDL from blood ii Dietary fiber binds bile acids 1 Prevents recycling requires constant bile acid synthesis from cholesterol by liver cells so more cholesterol used up VI Transferrin RME iii Cholesterol deficit in cell upregulates receptor synthesis turns on gene to make more LDLRs 1 More receptors increase LDL uptake clearance from blood a Ligand ferrotransferrin transferrin protein with Fe3 bound b Transferrin receptor binds ferrotransferrin at pH 7 0 c Late endosome low pH dissociates Fe3 from transferrin i Does not dissociate transferrin from receptor just Fe3 from ligand Apo prefix means no Fe3 d Apotransferrin recycled to PM bound to receptor e Apotransferrin released from receptor at pH 7 0 VII IgG RME Transcytosis of IgG a Low pH of intestinal lumen promotes binding of Fc domain of IgG ligand in mother s milk to Fc receptor FcRn in apical membrane of intestinal cell Internalization via clathrin mediated RME b c Late endosome low pH maintains receptor ligand interaction stays bound d Recovery recycling of receptor ligand complex sorts ligand from fluid phase material taken in nonspecficially i Fluid phase material destroyed in lysosome e Receptor ligand complex transcytosed to basolateral membrane pH 7pH 7 pH 5 6 pH 5 6 i Exposed to neutral pH blood disrupts receptor ligand interaction ii iii FcRn receptor recycled to apical membrane via RME IgG released from bloodstream f Receptor recycled and ligand transcytosed Macromolecular Transport Virus and Toxin RME Phagocytosis I Enveloped viruses a Viruses are not cells i Contain limited genome but lack components required for life such as ribosomes and mitochondria b Viruses are incapable of reproducing outside cells i Commandeer cellular machinery to reproduce c E g Influenza virus HIV virus rabies virus d Genome i DNA or RNA retroviruses have RNA e g HIV and influenza ii Small number of genes HIV RNA genome is 900 nucleotides long 9 genes e Virus enzymes i Reverse transcriptase Integrase ii iii Viral protease f Capsid g Virus envelope i Protein coat surrounding genome and few viral enzymes i Phospholipid bilayer membrane similar to cell PM 1 Buds from infected cell PM ii Contains numerous copies of a few types of proteins called coat proteins Influenza HA protein 1 2 HIV gp120 II Enveloped virus infection of cells via RME a 1 Binding i Virus coat protein binds to a