1、Chapter 6 Cytoplasmic matrix,Endomembrane system,Protein Sorting and membrane traffickingLearning objective1.Compartmentalization in Eukaryotic Cells;The structural and functional relationship among the ER,Golgi complexes,lysosomes and plasma membranes of eukaryotic cells;The pathways of proteins ta
2、rgeting and sorting,and its mechanisms;The ways of protein modifications and intracellular sites after they are synthesized;Types of vesicle transport and their functions.1.The Compartmentalization in Eukaryotic CellsMembranes divide the cytoplasm of eukaryotic cells into distinct compartments.Three
3、 categories in eukaryotic cells:(1)the endomembrane system:ER,Golgi complex,Lys.,secretory vesicles.(2)the cytosol.(3)mitochondria,chloroplasts,peroxisomes,and the nucleus.Membrane-bound structures(organelles)are found in all eukaryotic cells.vCytoplasmic matrix and its functionsCytoplasmic Matrix:T
4、he region of fluid content of the cytoplasm outside of the membranous organelles.Aqueous solution of large and small molecules including filaments of cytoskeleton which act as organizer for some order.The Cytosol is the site of protein synthesis and degradation or modification.It also performs most
5、of the cells intermediary metabolism.Cytoplasmic matrix(Cytosol)and Endomembrane SystemFunctions of cytoplasmic matrix:The protein synthesis,degradation and modification.Cells carefully monitor the amount of misfolded proteins.An accumulation of misfolded proteins in the cytosol triggers a heat-shoc
6、k response,which stimulates the transcription of genes encoding cytosolic chaperones that help to refold the proteins.B.Endomembrane SystemEndomembrane System:The structural and functional relationship organelles including ER,Golgi complex,lysosome,endosomes,secretory vesicles.Membrane-bound structu
7、res(organelles)are found in all eukaryotic cells.Intracellular compartment%of total cell volumeCytosol 54Mittchondria 22Rough ER cisternae 9Smooth ER cisternae plus Golgi cisternae 6Nucleus 6Peroxisome 1Lysosomes 1Endosomes 1Relative volumes occupied by the major intracellular compartments in Liver
8、CellC.The Dynamic Nature of the Endomembrane SystemMost organelles are part of a dynamic system in which vesicles move between compartments.Biosynthetic parthways move proteins,carbohydrates and lipids within the cell.Secretory pathways discharge proteins from cells.Endocytic parthways move material
9、s into cells.Sorting signals are recognized by receptors and target proteins to specific sites.D.A few approaches to the study of cytomembranes Insights gained from autoradiography;Insights gained from the biochemical analysis of subcellular fractions;Insights gained from the study of genetic mutant
10、s;The dynamic activities of endomembrane systems are highly conserved despite the structural diversity of different cell types.De Duve,A.Claude and G.Palade,1974 Nobel Plrize2.The structure and functions of Endoplasmic Reticulum(ER)Rough endoplasmic reticulum and Smooth endoplasmic reticulum RER has
11、 ribosomes on the cytosolic side of continuous,flattened sacs(cisternae);SER is an interconnecting network of tubular membrane elements.Microsome(100-200nm)rER of pancreatic cellsMicrosomes are heterogeneous mixtures of similar-sized vesicles,formed from membranes of the ER and Golgi complex.Microso
12、mes retain activity during purification,allowing studies of function and composition.A.Functions of the rERProteins synthesized on ribosomes of rER include:secretory proteins,integral membrane proteins,soluble proteins of organelles.Modification and processing of newly synthesized proteins:glycosyla
13、tion in the rER;N-linked:linked to the amide nitrogen of asparagine(ER)O-linked:linked to the hydroxyl group serine or threonine via GalNac(in Golgi)The precursor of 14 residues is the same in plants,animals,and single-celled eukaryotesthen remove 3 glucoses and 1 mannose in the ERProteins synthesiz
14、ed on ribosomes of rER include:Membrane-bound structures(organelles)are found in all eukaryotic cells.(1)One might suspect that they function to aid folding and the transport process;for example,carbohydrate as a marker during protein folding in ER and the use of carbohydrate-binding lectins in guid
15、ing ER-to-Golgi transport.The Functions of Golgi complexGlycosylation in the Golgi complexcisternal maturation model and vesicular transport modelO-linked oligosaccharides takes place in Golgi complex.gondii RH tachyzoites replicating by endodyogeny in HFF cells.O-linked:linked to the hydroxyl group
16、 serine or threonine via GalNac(in Golgi)嫁堪蚂亮唆石谜盂闸祸寒碎擂椅奢诱傣样觉榨圣垒咸爷僵庆杰媳舟晤争蹋考研资料：北京大学细胞生物学 第六章 基质与内膜(上)考研资料：北京大学细胞生物学 第六章 基质与内膜(上)(2)Limit the approach of other macromolecules to the protein surface,more resistant to digestion by proteases.Protein disulfide isomerase(PDI);Microsomes retain activity dur
17、ing purification,allowing studies of function and composition.Stable expression of mammalian Golgi proteins.Membrane-bound structures(organelles)are found in all eukaryotic cells.Quality control of of newly synthesized proteins-The role of N-linked glycosylation in ER protein foldingQuality control:
18、ensuring that misfolded proteins do not leave ERThe lumen of rER contains:Bip and calnexin(chaperones):that recognize and bind to unfolded or misfolded proteins and give them correct conformation;Protein disulfide isomerase(PDI);GT(glucosyl-transferase,monitoring enenzyme)recognize unfolded or misfo
19、lded proteins and adds a glucose to the end of oligo.Synthesis of membrane lipidsMost membrane lipids are synthesized enterly within the ER.There are two exceptions:sphingomyelin and glycolipids,(begins in ER;completed in Golgi);(2)some of the unique lipids of the Mit and Chl membranes(themself).The
20、 membranes of different 0rganelles have markedly different lipids composition.Transport by budding：ERGC、Ly、PMTransport by phospholipid exchange proteins(PEP)：ERother organelles（including Mit and Chl）d,Quantification of images in a and b.Cells carefully monitor the amount of misfolded proteins.Protei
21、n disulfide isomerase(PDI);The Golgi networks are processing and sorting stations where proteins are modified,segregated and then shipped in different directions.Rough ER cisternae 9Insights gained from the study of genetic mutants;布吸浑辆究砚捶寇佬幢冒扭藐累罕捕墟强趴阔农葛针屠钮敝屉掉武孪宽膊考研资料：北京大学细胞生物学 第六章 基质与内膜(上)考研资料：北京大学
22、细胞生物学 第六章 基质与内膜(上)The ways of protein modifications and intracellular sites after they are synthesized;Smooth ER cisternae plus Golgi cisternae 6Endomembrane SystemThe role of phospholipid translocators in lipid bilayer synthesisModification and processing of newly synthesized proteins:glycosylation
23、 in the rER;诬麓销沫厨瞒瞄耳埔迹卧挫堪扫回览厨侈弃刨魏簇造波屏霖慧页呸雄皇严考研资料：北京大学细胞生物学 第六章 基质与内膜(上)考研资料：北京大学细胞生物学 第六章 基质与内膜(上)Types of vesicle transport and their functions.d,Quantification of images in a and b.Stable expression of mammalian Golgi proteins.It also performs most of the cells intermediary metabolism.The role of
24、phospholipid translocators in lipid bilayer synthesisphospholipid translocators=Scramblase(ABC transporter Family)B.Functions of the sERSynthesis of steroids in endocrine cells.Detoxification of organic compounds in liver cells.System of oxygenases-cytochrome p450 familyRelease of glucose 6-phosphat
25、e in liver cells.Sequestration of Ca2+.Ca2+-ATPase3.The structure and functions of Golgi complexA.The polarity of Golgi complexa)Cis cisternae of Golgi complex:reduced osmium tetroxide(OsO4);b)Reaction for enzyme mannosidase II,localized in the medial;c)Reaction for enzyme nucleoside diphosphatase,l
26、ocalized in the trans cisternae.Regional differences in membrane composition across the Golgi stackB.The Functions of Golgi complexGlycosylation in the Golgi complexGolgi complex plays a key role in the assembly of the carbohydrate component of glycoproteins and glycolipids.The core carbohydrate of
27、N-linked oligosaccharides is assembled in the rER.Modifications to N-linked oligosaccharides are completed in the Golgi complex.O-linked oligosaccharides takes place in Golgi complex.Structure of typical O-and N-linked oligosaccharidesCore RegionAfter R.Kornfeld and S.Kornfeld,1985,Annu.Rev.Biochem.
28、45:631What is the purpose of glycosylation?N-linked glycosylation is prevalent in all eucaryotes,but is absent from procaryotes.It dont require a template.There is an important difference between the construction of an oligosaccharide and the synthesis of DNA,RNA,and protein.Important functions:(1)O
29、ne might suspect that they function to aid folding and the transport process;for example,carbohydrate as a marker during protein folding in ER and the use of carbohydrate-binding lectins in guiding ER-to-Golgi transport.(2)Limit the approach of other macromolecules to the protein surface,more resist
30、ant to digestion by proteases.(3)Regulatory roles in signaling through the cell-surface receptor Notch,to allows these cells to respond selectively to activating stimuli.The Golgi networks are processing and sorting stations where proteins are modified,segregated and then shipped in different direct
31、ions.Golgi complex and cells secretionContinual,unregulated discharge of material from the cellsThe discharge of products stored in cytoplasmic granules,in response to appropriate stimuli.Vesivular transport within the Golgi apparatus:Two views:cisternal maturation model and vesicular transport mode
32、lTwo possible models explaining the organization of the Golgi complex and the transport from one cisterna to the next.十 十 十 C.Golgi BiogenesisStages of Golgi growth and division.Shown are thin section electron micrographs of T.gondii RH tachyzoites replicating by endodyogeny in HFF cells.Cells were
33、placed in one of four categories according to the number and size of the Golgi:a,single Golgi;b,single,elongated Golgi;c,two Golgi;d,Golgi,often more vesiculated,ineach nascent daughter cell,delineated by the growing inner membrane complex(IMC).a,apicoplast;dg,dense granules;er,ER;es,ER exit sites o
34、n the outer flattened part of the nuclear envelope;G,Golgi;m,micronemes;mit,mitochondria;r,rhoptries.Scale bar,0.5mm.Stable expression of mammalian Golgi proteins.a,b,Overlaid immunofluorescence and phase images of GRASPYFP(a)and NAGTIYFP(b)in stable,transgenic cell lines of Toxoplasma gondii.ch,Imm
35、unofluorescence images of a transgenic cell line expressing both GRASPCFP(green)and NAGTIYFP(red)before(ce)or after(fh)treatment with 5mg/ml BFA for 10 min at 37C.Merged images are shown on the right.Asterisks indicate a secreted form of NAGTIYFP that accumulates in the parasitophorous vacuole.Scale
36、 bars,5mm.Immunoelectron microscopy of transgenic parasites.ac,Cryosections of GRASPYFP(a,c)or NAGTIYFP(b)transgenic parasites,pretreated for 2 h with 50mg/ml cycloheximide,before being fixed and immunolabelled for YFP using polyclonal antibodies against GFP followed by protein A coupled to 5-nm gol
37、d particles.Note the high density of labelling restricted to Golgi membranes.In c,GRASPYFP transgenic parasites were treated with BFA(5mg/ml)for 30 min before immunolabelling.Note the tubulo-vesicular appearance of the Golgi caused by loss of Golgi enzymes to the ER.d,Quantification of images in a and b.Results are presented as mean s.d.gold particles/um2.
