1、1.The Compartmentalization in Eukaryotic CellsvMembranes divide the cytoplasm of eukaryotic cells into distinct compartments.Three categories in eukaryotic cells:(1)the endomembrane system:ER,Golgi complex,Lys.,secretory vesicles.(2)the cytosol.(3)mitochondria,chloroplasts,peroxisomes,and the nucleu
2、s.vMembrane-bound structures(organelles)are found in all eukaryotic cells.D.A few approaches to the study of cytomembranesv Insights gained from autoradiography;v Insights gained from the biochemical analysis of subcellular fractions;vInsights gained from the study of genetic mutants;The dynamic act
3、ivities 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 ribosomes on the
4、 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.Microsomes retain activi
5、ty during purification,allowing studies of function and composition.A.Functions of the rERv Proteins synthesized on ribosomes of rER include:secretory proteins,integral membrane proteins,soluble proteins of organelles.v Modification and processing of newly synthesized proteins:glycosylation in the r
6、ER;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 ERv Quality control of of newly s
7、ynthesized proteins-The role of N-linked glycosylation in ER protein foldingQuality control: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 disu
8、lfide isomerase(PDI);GT(glucosyl-transferase,monitoring enenzyme)recognize unfolded or misfolded proteins and adds a glucose to the end of oligo.vSynthesis of membrane lipids Most membrane lipids are synthesized enterly within the ER.There are two exceptions:sphingomyelin and glycolipids,(begins in
9、ER;completed in Golgi);(2)some of the unique lipids of the Mit and Chl membranes(themself).The 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）The rol
10、e of phospholipid translocators in lipid bilayer synthesisphospholipid translocators=Scramblase(ABC transporter Family)B.Functions of the sERvSynthesis of steroids in endocrine cells.vDetoxification of organic compounds in liver cells.System of oxygenases-cytochrome p450 familyvRelease of glucose 6-
11、phosphate in liver cells.vSequestration 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 diphos
12、phatase,localized in the trans cisternae.vRegional differences in membrane composition across the Golgi stackB.The Functions of Golgi complexvGlycosylation in the Golgi complexGolgi complex plays a key role in the assembly of the carbohydrate component of glycoproteins and glycolipids.The core carbo
13、hydrate of 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.R
14、ev.Biochem.45:631vWhat 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 fu
15、nctions:(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 guiding ER-to-Golgi transport.(2)Limit the approach of other macromolecules to the protein surface
16、,more resistant 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.vThe Golgi networks are processing and sorting stations where proteins are modified,segregated and then shipped in dif
17、ferent directions.vGolgi complex and cells secretionContinual,unregulated discharge of material from the cellsThe discharge of products stored in cytoplasmic granules,in response to appropriate stimuli.v Vesivular transport within the Golgi apparatus:Two views:cisternal maturation model and vesicula
18、r transport modelTwo 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 c
19、ells.Cells were 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
20、,ER exit sites on 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 Toxoplas
21、ma gondii.ch,Immunofluorescence 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 parasitophoro
22、us vacuole.Scale 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 cou
23、pled to 5-nm gold 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.
