Transcript Golgi
Golgi complex, secretion and protein transport Biology I – Faculty of Pharmacy Dr. Eszter Lajkó Department of Genetics, Cell- and Immunobiology 03.10.2016 Protein targeting and sorting Transport of proteins from the synthesis site to their destinations “Roadmap” of protein traffic Key components of the protein transport 1. Sorting signal 2. Receptors: recognize sorting signal and guide proteins to their appropriate destination 3. Way of protein transfer • Gated transport through the nuclear pore • Translocation across the membrane (transmembrane protein traslocator = translocon) • Vesicular transport 4. Energy Protein sorting Protein synthesis in cytoplasm (free ribosome) Co-translational transmembrane transport Endoplasmic reticulum (membrane-bounded ribosome) (hydrophobic aa at N-term) Gated transport Post-translational transmembrane transport Cytoplasm (no signal) Vesicular transport Golgi Nucleus (NLS) Late endosome Mitochondria (N-term. positively charged aa) Peroxisomes Lysosome Early endosome Secretory vesicle (3 aa at C-term) Plasma membrane Vesicular transport • transport between membrane-enclosed compartments • transport of macromolecules (soluble and membrane-bound) from the donor compartment to the target compartment Further information about the vesicular transport in 6th week lecture Main vesicular transport pathways Inward transport Endocytotic pathway Outward transport Secretory pathway Golgi – „Traffic manager” of the cell Golgi apparatus "internal reticular apparatus" Camillo Golgi (1843-1926) Nobel prize 1906 Metal impregnation Structure of Golgi apparatus • consist of saccules (cisternae), tubules and vesicles • structural-functional unit: dictyosome (4-6 saccules) • structure is polarized into sub-compartments Endoplasmic reticulum entry Cis Golgi network (CGN) Cis Golgi (CG) Medial Golgi (MG) Trans Golgi (TG) Trans Golgi network (TGN) exit Plasma membrane Lysosome Position Nucleus Microtubule Golgi Role of microtubules in maintance of Golgi structure Intact microtubules Disintegration of microtubules Golgi- green Microtubules - red 2002 by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. Visualisation of Golgi with different techniques Silver impregnation for LM TEM 1. Golgi dyctiosomes Multiphoton fluorescence image Main functions Sructural and functional polarization Cis face: arrival side 1. post-translational modifications of proteins (and lipids) 2. sorting 3. formation of transport vesicles 4. transport trans face: departure side Vesicular tubular cluster • • • • continually generated from ER-derived vesicles separate compartment from ER and Golgi transport container moving along the microtubules to the Golgi Cis Golgi Network – Entry side • collection of fused vesicular tubular clusters • the proteins and lipids arrive from the ER in vesicles ER-resident protein move back to the ER proteins of Golgi targeting to the cell surface or another compartments they are N-glycosylated no sorting in the ER move to the cis Golgi cisterna Glycosylation • Adding oligosaccharide chain to protein (lipids) N-glycosylation O-glycosylation • Made by glycosyl transferases soluble glycoportein • N-glycosylation (to Asn) is made in ER and modified in ER and Golgi (longer chains) O-glycosylation Glycolipid N-glycosylation • O-glycosylation (to Ser, Thr) is made in Golgi (shorter chains) Membrane glycoportein N-glycosylation • Preformed oligosaccharide chain is transfered to asparagine side chains of a polypeptide in ER • Processing of oligosaccharide chain starts in ER and continues in Golgi cytoplasm Asparagine side chain ER lumen Golgi: further processing of N-linked olgosaccharide chain Modification of N-oligosaccharide chains of proteins 1. phosphorylation of the mannoses of the lysosomal proteins - cis Golgi network 2. no change (high mannose) - cis Golgi 3. change of mannoses to other monosaccharides - medial and trans Golgi • each cisternae containing a characteristic mixture of enzymes involved in processing of N-linked oligosaccharides • enzymes are all membrane bound • processing depends on the position of oligosaccharides in the protein Sorting and modification of lysosomal enzymes Mannose-6-phosphate (M-6-P) signal • based on the recognition of lysosomal hydrolases • recognition of the “signal patches” is required • main working enzyme: GlcNAc-phosphotransferase Lysosomal enzymes get a M-6-P signal in CGN Significance of M-6-P labelling Lysosomal enzyme Lysosome Mannose-6phosphate M-6-P Lysosomal enzymes with M-6-P signal are - not modified further in Golgi - recognised by receptors in trans Golgi network - transported to the lysosome Golgi Main types of N-oligosaccharide chains -Asn -Asn -Asn Steps of processing and subsequent sugar addition is rigidly ordered, but the complex oligosaccharides can be heterogeneous cis Golgi medial Golgi medial Golgi trans Golgi negatively charged trans Golgi N- and O-linked glycosylation Threonine, Serine O-linked glycosylation -Ser/Thr -Ser/Thr • • • • takes place mainly in the medial- and trans Golgi shorter oligosaccharide chains adding monosaccharides one by one to Ser/Thr each step requires different enzymes Significance of glycosylation • folding • sorting • protection against proteolytic enzymes • makes proteins hydrophylic • cell adhesion (leukocytes and endothel – cell adhesion molecules) • antigenity (A,B,O blood groups) • glycocalyx (external coat) Proteoglycan synthesis Basic structure of proteoglycans Serine linker: tetrasaccharide Glycosaminoglycan (GAG) core protein • Proteins with polisacharide side chains (long, unbranched) • Found in plasmambrane and extracellular matrix • Major components: glycosaminoglycan (GAG) - composed of repated disaccharides - containing sulphate group negatively charged - e.g. hyaluronic acid, chondroitin sulphate, dermatan sulphate, heparan sulphate Proteoglycans N and O oligosaccharides are not shown Modifications of proteins and lipids in Golgi 1. Glycosylation • modification of oligosaccharide chains of proteins, • binding of newly synthesized oligo- or polysaccharide chains - M-6-P group (lysosomal proteins) - no change (high mannose) - change of mannoses to other monosaccharides - O-glycosylation - proteoglycans synthesis 2. adding of -SO4 group - carbohydrates: GAG, proteoglycans - amino acid: Tyr residue of proteins 3. synthesis of lipids - glycolipids - sphingomyelin 4. proteolysis chondroithine sulphate Synthesis of membrane lipids Golgi Sphingomyelin ER Monosaccharide Clycolipid Cerebroside Oligosaccharide Gangliosode A,B,0 blood group antigens (glycolipids = ganglioside) ceramide sugar residues Golgi is a major protein sorting • back to ER - ER resident proteins (soluble) – KDEL signal cis Golgi network - membrane proteins – KKXX signal and from all cisternae cargo receptors proteins needed for vesicule formation and fusion • retaining Golgi proteins - aggregation of Golgi proteins in all cisternae • to lysosome - M-6-P signal • to plasmamembrane and ECM • to cell exterior (for secretion) trans Golgi network Main pathways going in and out Golgi Bidirectional transport between ER and Golgi Vesicular tubular cluster Secretory protein ER-resident protein receptor (KDEL receptor) ER-resident protein with KDEL signal sequence Transport through the Golgi apparatus Cisternal maturation model Vesicle transport model • Cisternae mature from early to late by acquiring and then losing specific Golgi-resident proteins • Cisternae remain at the same place with characteristic set of Golgi proteins • Cisternae moves through the dictyosome with cargo in its lumen • Cargo protein are moved forward by transport vesicle • Retrograde transport of Golgi enzymes • Retrograd pathway of vesicles return the escaped proteins to the previous cisternae Golgi resident proteins - enzyme-content of the different compartments Unstained cis face trans face cis Golgi medial Golgi trans Golgi mannosidase I mannosidase II nucleoside diphosphatase Osmium impregnation Bidirectional transport of proteins Forward – anterograd transport • lysosomal enzymes • secretory proteins • extracellular matrix and plasma membrane components • Golgi-resident proteins Backward – retrograd transport • proteins of the ER recycled - membrane-bound or soluble - retention signal is required • protein of each cisterna • M-6-P receptor from the lysosomes • membrane components of transport vesicles from the plasma membrane Main transport pathways from TGN Proteins are seggregated into different transport packages and dispatched exocytosis transport from the TGN to the cell exterior secretory vesicles secretion synthesis, modification and release (exocytosis) of different compounds (e.g. proteins, lipids) • central organelles – ER and Golgi • types: consitutive secretion regulated secretion (fusion of transport vesicles with plasma membrane) lysosome endosomal-lysosomal compartment Constitutive secretion (exocytosis) • • • • • • signal default pathway presents in all cells continuously non-selective no accumulation of vesicles ECM proteins, membrane lipids and proteins Regulated secretion (exocytosis) signal • typical for glandular cells and neurons • signal is needed for the exocytosis • accumulation of vesicles • hormones, eurotransmitters, digestive enzymes Pathways of protein sorting in the TGN Modifications of secretory vesicles • selective aggregation - TGN • further modifications and sorting - inactive precursor - active enzyme or hormone (e.g. preproinsulin - proinsulin - insulin) • concentration - loss of water • hydratation - e.g. proteoglygans • uptake some cytoplasmatic substances e.g. histamine Immature secretory vesicle contains missorted proteins Removal of missorted proteins Acidification and condensation Proteolysis of proteins in secretory vesicles proteolysis pro-hormone inactive hormone active Synthesis of pre-pro-hormone inactive Glycolistaion of pro-hormone inactive Secretory vesicle with pro-hormone inactive active hormone Network of membran flow in eukaryotic cells