1 Golgi complex, secretion and protein transportBiology I – Faculty of Pharmacy Dr. Eszter Lajkó Department of Genetics, Cell- and Immunobiology

2 Protein targeting and sortingTransport of proteins from the synthesis site to their destinations Overview of sorting of nuclear-encoded proteins in eukaryotic cells. All nuclear-encoded mRNAs are translated on cytosolic ribosomes. Ribosomes synthesizing nascent proteins in the secretory pathway 1 are directed to the rough endoplasmic reticulum (ER) by an ER signal sequence 2 . After translation is completed in the ER, these proteins move via transport vesicles to the Golgi complex dlccirc3; from whence they are further sorted to several destinations 4a, 4b, 4c . After synthesis of proteins lacking an ER signal sequence is completed on free ribosomes 1 , the proteins are released into the cytosol 2 . Those with an organelle- specific uptake-targeting sequence are imported into the mitochondrion 3a , chloroplast 3b , peroxisome 3c , or nucleus 3d . Mitochondrial and chloroplast proteins typically pass through the outer and inner membranes to enter the matrix or stromal space, respectively. Some remain there, and some 4a are sorted to other organellar compartments. Unlike mitochondrial and chloroplast proteins, which are imported in a partially unfolded form, most peroxisomal proteins cross the peroxisome membrane as fully folded proteins 4b . Folded nuclear proteins, often in the form of ribonucleoprotein particles, enter through visible nuclear pores by processes discussed in Chapter 11 4c.

3 “Roadmap” of protein traffic

4 Key components of the protein transportSorting signal Receptors: recognize sorting signal and guide proteins to their appropriate destination Way of protein transfer Gated transport through the nuclear pore Translocation across the membrane (transmembrane protein traslocator = translocon) Vesicular transport Energy

5 (hydrophobic aa at N-term) (N-term. positively charged aa)Protein sorting Co-translational transmembrane transport Protein synthesis in cytoplasm (free ribosome) Endoplasmic reticulum (membrane-bounded ribosome) (hydrophobic aa at N-term) Gated transport Vesicular transport Cytoplasm (no signal) Post-translational transmembrane transport Golgi Nucleus (NLS) Late endosome Secretory vesicle Mitochondria (N-term. positively charged aa) Lysosome Early endosome Peroxisomes (3 aa at C-term) Plasma membrane

6 Further information about the vesicular transport in 6th week lecturetransport 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

7 Main vesicular transport pathwaysInward transport Endocytotic pathway Outward transport Secretory pathway

8 Golgi – „Traffic manager” of the cell

9 Golgi apparatus "internal reticular apparatus" Camillo Golgi( ) Nobel prize 1906 Metal impregnation

10 Structure of Golgi apparatusconsist of saccules (cisternae), tubules and vesicles structural-functional unit: dictyosome (4-6 saccules) structure is polarized into sub-compartments Endoplasmic reticulum Cis Golgi network (CGN) Cis Golgi (CG) Medial Golgi (MG) Trans Golgi (TG) Trans Golgi network (TGN) entry exit Plasma membrane Lysosome

11 Position Nucleus Microtubule Golgi

12 Role of microtubules in maintance of Golgi structureIntact microtubules Disintegration of microtubules Golgi- green Microtubules - red 2002 by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.

13 Visualisation of Golgi with different techniquesSilver impregnation for LM TEM 1. Golgi dyctiosomes Multiphoton fluorescence image

14 Main functions Sructural and functional polarizationCis face: arrival side post-translational modifications of proteins (and lipids) sorting formation of transport vesicles transport trans face: departure side

15 Vesicular tubular clustercontinually generated from ER-derived vesicles separate compartment from ER and Golgi transport container moving along the microtubules to the Golgi

16 Cis Golgi Network – Entry sidecollection 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 move to the cis Golgi cisterna they are N-glycosylated no sorting in the ER

17 Glycosylation Adding oligosaccharide chain to protein (lipids)N-glycosylation Adding oligosaccharide chain to protein (lipids) Made by glycosyl transferases N-glycosylation (to Asn) is made in ER and modified in ER and Golgi (longer chains) O-glycosylation (to Ser, Thr) is made in Golgi (shorter chains) O-glycosylation soluble glycoportein O-glycosylation Glycolipid N-glycosylation Membrane glycoportein

18 further processing of N-linked olgosaccharide chainN-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

19 Modification of N-oligosaccharide chains of proteinsphosphorylation of the mannoses of the lysosomal proteins cis Golgi network no change (high mannose) cis Golgi 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

20 Sorting and modification of lysosomal enzymesMannose-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

21 Lysosomal enzymes get a M-6-P signal in CGN

22 Significance of M-6-P labellingLysosomal enzyme Lysosome Mannose-6-phosphate 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

23 Main types of N-oligosaccharide chains-Asn -Asn -Asn

24 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

25 N- and O-linked glycosylationThreonine, Serine

26 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

27 Significance of glycosylationfolding 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)

28 Proteoglycan synthesisBasic 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

29 Proteoglycans N and O oligosaccharides are not shown

30 Modifications of proteins and lipids in GolgiGlycosylation 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 adding of -SO4 group carbohydrates: GAG, proteoglycans amino acid: Tyr residue of proteins synthesis of lipids glycolipids sphingomyelin proteolysis chondroithine sulphate

31 Synthesis of membrane lipidsGolgi Sphingomyelin ER Monosaccharide Oligosaccharide Cerebroside Clycolipid Gangliosode

32 A,B,0 blood group antigens(glycolipids = ganglioside) ceramide sugar residues

33 Golgi is a major protein sortingback to ER ER resident proteins (soluble) – KDEL signal membrane proteins – KKXX signal cargo receptors proteins needed for vesicule formation and fusion retaining Golgi proteins aggregation of Golgi proteins to lysosome M-6-P signal to plasmamembrane and ECM to cell exterior (for secretion) cis Golgi network and from all cisternae in all cisternae trans Golgi network

34 Main pathways going in and out Golgi

35 Bidirectional transport between ER and GolgiER-resident protein receptor (KDEL receptor) ER-resident protein with KDEL signal sequence Secretory protein Vesicular tubular cluster

36 Transport through the Golgi apparatusCisternal maturation model Vesicle transport model Cisternae mature from early to late by acquiring and then losing specific Golgi-resident proteins Cisternae moves through the dictyosome with cargo in its lumen Retrograde transport of Golgi enzymes Cisternae remain at the same place with characteristic set of Golgi proteins Cargo protein are moved forward by transport vesicle Retrograd pathway of vesicles return the escaped proteins to the previous cisternae

37 Golgi resident proteins - enzyme-content of the different compartments - cis Golgi mannosidase I medial Golgi mannosidase II trans Golgi nucleoside diphosphatase Unstained cis face trans face Osmium impregnation

38 Bidirectional transport of proteinsForward – 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

39 Main transport pathways from TGNProteins are seggregated into different transport packages and dispatched exocytosis transport from the TGN to the cell exterior (fusion of transport vesicles with plasma membrane) 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 lysosome endosomal-lysosomal compartment

40 Constitutive secretion (exocytosis)default pathway presents in all cells continuously non-selective no accumulation of vesicles ECM proteins, membrane lipids and proteins signal

41 Regulated secretion (exocytosis)typical for glandular cells and neurons signal is needed for the exocytosis accumulation of vesicles hormones, eurotransmitters, digestive enzymes signal

42 Pathways of protein sorting in the TGN

43 Modifications of secretory vesiclesselective 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

44 Immature secretory vesicle contains missorted proteinsRemoval of missorted proteins Acidification and condensation

45 Proteolysis of proteins in secretory vesiclesSecretory vesicle with pro-hormone inactive Glycolistaion of pro-hormone hormone active pro-hormone Synthesis of pre-pro-hormone proteolysis

46 Network of membran flow in eukaryotic cells