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1、Chapter 6,Cytoplasmic matrix, Endomembrane system, Protein Sorting and membrane trafficking,Learning objective,1. Compartmentalization in Eukaryotic Cells; The structural and functional relationship among the ER, Golgi complexes, lysosomes and plasma membranes of eukaryotic cells; The pathways of pr
2、oteins targeting 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 Cells,Membranes divide the cytoplasm of eukaryotic cells into distinct com
3、partments. Three 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.,Cytoplasmic matrix and i
4、ts functions,Cytoplasmic Matrix: The 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
5、 modification. It also performs most of the cells intermediary metabolism.,Cytoplasmic matrix (Cytosol) and Endomembrane System,Functions of cytoplasmic matrix:,The protein synthesis, degradation and modification.,Cells carefully monitor the amount of misfolded proteins. An accumulation of misfolded
6、 proteins in the cytosol triggers a heat-shock response, which stimulates the transcription of genes encoding cytosolic chaperones that help to refold the proteins.,B. Endomembrane System,Endomembrane System : The structural and functional relationship organelles including ER,Golgi complex, lysosome
7、, endosomes, secretory vesicles. Membrane-bound structures (organelles) are found in all eukaryotic cells.,Relative volumes occupied by the major intracellular compartments in Liver Cell,C. The Dynamic Nature of the Endomembrane System,Most organelles are part of a dynamic system in which vesicles m
8、ove between compartments. Biosynthetic parthways move proteins, carbohydrates and lipids within the cell. Secretory pathways discharge proteins from cells. Endocytic parthways move materials into cells. Sorting signals are recognized by receptors and target proteins to specific sites.,D. A few appro
9、aches 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 mutants; The dynamic activities of endomembrane systems are highly conserved despite the structural diversity of
10、 different cell types.,De Duve, A.Claude and G.Palade,1974 Nobel Plrize,2. The structure and functions of Endoplasmic Reticulum(ER),Rough endoplasmic reticulum and Smooth endoplasmic reticulum,RER has ribosomes on the cytosolic side of continuous, flattened sacs(cisternae); SER is an interconnecting
11、 network of tubular membrane elements.,Microsome(100-200nm),rER of pancreatic cells,Microsomes are heterogeneous mixtures of similar-sized vesicles, formed from membranes of the ER and Golgi complex. Microsomes retain activity during purification, allowing studies of function and composition.,A. Fun
12、ctions of the rER,Proteins synthesized on ribosomes of rER include: secretory proteins, integral membrane proteins, soluble proteins of organelles.,Modification and processing of newly synthesized proteins: glycosylation in the rER;,N-linked: linked to the amide nitrogen of asparagine (ER) O-linked:
13、 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 eukaryotes,then remove 3 glucoses and 1 mannose in the ER,Quality control of of newly synthesized proteins-The role of N-linked glycosylation in ER p
14、rotein folding,Quality control: ensuring that misfolded proteins do not leave ER,The 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, mon
15、itoring enenzyme ) recognize unfolded or misfolded proteins and adds a glucose to the end of oligo,Synthesis of membrane lipids,Most 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 uni
16、que lipids of the Mit and Chl membranes (themself). The membranes of different 0rganelles have markedly different lipids composition. Transport by budding：ERGC、Ly、PM Transport by phospholipid exchange proteins(PEP)：ERother organelles（including Mit and Chl）,The role of phospholipid translocators in l
17、ipid bilayer synthesis,phospholipid translocators = Scramblase (ABC transporter Family),B. Functions of the sER,Synthesis of steroids in endocrine cells. Detoxification of organic compounds in liver cells. System of oxygenases-cytochrome p450 family Release of glucose 6-phosphate in liver cells. Seq
18、uestration of Ca2+. Ca2+-ATPase,3. The structure and functions of Golgi complex,A.The polarity of Golgi complex,a) 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 , locali
19、zed in the trans cisternae.,Regional differences in membrane composition across the Golgi stack,B. The Functions of Golgi complex,Glycosylation in the Golgi complex,Golgi complex plays a key role in the assembly of the carbohydrate component of glycoproteins and glycolipids.,The core carbohydrate of
20、 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 oligosaccharides,Core Region,After R. Kornfeld and S. Kornfeld, 1985, Annu.
21、Rev. Biochem. 45:631,What 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. Impo
22、rtant functions: (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 p
23、rotein surface, 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.,The Golgi networks are processing and sorting stations where proteins are modified, segregated and
24、then shipped in different directions.,Golgi complex and cells secretion,Continual,unregulated discharge of material from the cells,The discharge of products stored in cytoplasmic granules, in response to appropriate stimuli.,Vesivular transport within the Golgi apparatus: Two views: cisternal matura
25、tion model and vesicular transport model,Two possible models explaining the organization of the Golgi complex and the transport from one cisterna to the next.,十,十,十,C. Golgi Biogenesis,Stages of Golgi growth and division. Shown are thin section electron micrographs of T. gondii RH tachyzoites replic
26、ating by endodyogeny in HFF cells. 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, in each nascent daughter cell, delineated by the growing inner membrane complex (
27、IMC). a, apicoplast; dg, dense granules; er, ER; es, 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 GRA
28、SPYFP (a) and NAGTIYFP (b) in stable, transgenic cell lines of Toxoplasma 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.
29、Asterisks indicate a secreted form of NAGTIYFP that accumulates in the parasitophorous 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 fi
30、xed and immunolabelled for YFP using polyclonal antibodies against GFP followed by protein A coupled 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.,