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

2. Protein targeting and sorting
Transport 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 transport
Sorting 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 lecture
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

7. Main vesicular transport pathways
Inward 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 apparatus
consist 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 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.

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

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

15. Vesicular tubular cluster
continually 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 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
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 chain
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

19. Modification of N-oligosaccharide chains of proteins
phosphorylation 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 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

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

22. Significance of M-6-P labelling
Lysosomal 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 glycosylation
Threonine, 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 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)

28. 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

29. Proteoglycans
N and O oligosaccharides are not shown

30. Modifications of proteins and lipids in Golgi
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
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 lipids
Golgi
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 sorting
back 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 Golgi
ER-resident protein receptor
(KDEL receptor)
ER-resident protein with KDEL signal sequence
Secretory protein
Vesicular tubular cluster

36. 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 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 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

39. Main transport pathways from TGN
Proteins 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 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

44. Immature secretory vesicle contains missorted proteins
Removal of missorted proteins
Acidification and condensation

45. Proteolysis of proteins in secretory vesicles
Secretory 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