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1、B. Signal Hypothesis -G.Blobel & D.Sabatini,1971. A model for the Signal Mechanism of Cotranslational Import,Evidence That Protein Synthesized on Ribosomes Attached to ER Membranes Pass Directly into the ER Lumen (D.Sabatini) Milstein : IgG,Milstein et al: Studying the synthesis of light chain of Ig
2、G (in cell-free systems, 20 Aa longer at N-terminal end than the authentic light chain ),Adding ER membranes to this system leads to the production of an IgG light chain of the correct size.,A Schematic model for the synthesis of a secretory protein on a membrane-bound ribosome of the rough ER,Signa
3、l-recognition particle, SRP: Six different polypeptides complexed with a 300-nucleotide (7S)molecule of RNA. ER signal sequence: Typically 15-30 amino acids: Consist of three domains: a positively charged N-terminal region, a central hydrophobic region, and a polar region adjoining the site where cl
4、eavage from the mature protein will take place. A signal sequence on nascent seretory proteins targets them to the ER and is then cleaved off SRP receptor (GTP binding protein),SRP have three main active sites: One that recognizes and binds to ER signal sequence; One that interacts with the ribosome
5、 to block further translation; One that binds to the ER membrane (docking protein),The sorting signal of VSV glycoproteins : Asp-X-Gln或DXE,Figure 6-43. The sorting of proteins destined for the apical and basolateral plasma membranes of epithelial cells. When cultured MDCK cells are infected simultan
6、eously with VSV and influenza virus, the VSV glycoprotein is found only on the basolateral membrane, whereas the HA glycoprotein of the influenza virus is found only on the apical membrane. Like these viral proteins, some cellular proteins are sorted directly to the apical membrane and others to the
7、 basolateral membrane via specific transport vesicles that bud from the trans-Golgi network. In certain other polarized cells, some apical and basolateral proteins are transported together to the basolateral surface; the apical proteins then move selectively, by endocytosis and transcytosis, to the
8、apical membrane. K. Simons et al., Cell 62:207; K. Mostov et al., JCB. 116:577,Start-transfer Sequence & Stop-transfer Sequence,Figure 6-24. Synthesis and insertion into the ER membrane of the GLUT1 glucose transporter and other proteins with multiple transmembrane a-helical segments. The N-terminal
9、 a helix functions as an internal, uncleaved signal-anchor sequence (red), directing binding of the nascent polypeptide chain to the rER membrane and initiating cotranslational insertion. Both SRP and the SRP receptor are involved in this step. Following synthesis of helix 2, which functions as a st
10、op-transfer membrane-anchor sequence, extrusion of the chain through the translocon into the ER lumen ceases. The first two a helices then move out of the translocon into the ER bilayer, anchoring the nascent chain as an a-helical hairpin. The C-terminus of the nascent chain continues to grow in the
11、 cytosol. Subsequent a-helical hairpins could insert similarly, although SRP and the SRP receptor are required only for insertion of the first signalanchor sequence. Although only six transmembrane a helices are depicted here, GLUT1 and proteins of similar structure have twelve or more. H. P. Wessel
12、s et al., Cell 55:61.,The Orientation of Nascent Polypeptide,The Nascent polypeptide is oriented within translocon so that the positively charged flanking sequence faces the cytosol,C. A Model for the Postranslational Import of Polypeptides into the Mit.,Post-translational modification and quality c
13、ontrol in the rER,Disulfide bonds are formed and rearranged in the ER lumen Only in ER lumen is there a redox environment for oxidation of SH groups. PDI: protein disulfide isomerase, found in abundance in the ER lumen,Correct folding of newly made proteins is facilitated by several ER proteins,Prot
14、eins without any signal sequence are cytosol residual proteins,6. Types of Vesicle Transport and Their Functions,A. The three different types of coated vesicles. Different coat proteins select different cargo and shape the transport vesicles that mediate the various steps in the biosynthetic-secreto
15、ry and endocytic pathways.,COPII-coated vesicles move materials from the ER to the Golgi.,The assembly of a COPII-coated vesicles. SarGTP binding protein: Sar-GTP binds to the ER; Sar-GDP dissociates from the ER,Antibodies is able to block the budding of vesicle from ER but have no effect on vesicle
16、 transport from one Golgi compartment to another in mammalian cell.,COPI-coated vasicles transporting Escaped ER resident Proteins Back to the ER. The assembly of a COPI-coat is mediated by ADP-ribosylation factor(ARF), GTP binding protein, which is required for vesicle transfer between cisternae. C
17、OPI coated vesicles may select specific cargo. ER is an open prison. Soluble ER protein bear Retrieving signalKDEL(Lys-Asp-Glu-Leu)in mammal and HDEL in yeast, whereas ER membrane proteins bear the signal KKXX .The KDEL receptor present in vesicular tubular clusters and the Golgi apparatus (3) COPI-
18、coated vesicle were first identified by treatment of GTP analogues - COPI-coated vesicle accumulated within the cell and could be isolated by centrifugation.,A model for the retrieval of ER resident proteins. The KDEL receptor captures the soluble ER resident proteins and carries them in COPI-coated
19、 transport vesicles back to the ER. Neutral pH: dissociate from the KDEL; low pH: binding the KDEL,Clathrin-coated vesicle: Transporting Cargo from the TGN to endosomes, Lysosomes, and plant vacuoles and also move materials from the PM to cytoplasmic compartments along the endocytic pathway. The TGN
20、 of Golgi is the Sourse of Clathrin-coated vesicle. (2) Clathrin-coats contain: protein clathrin-which forms a structural scaffold, adaptors- multisubunit,which forms an inner shell.,The formation of clathrin-coated pits in the TGN,B. The SNARE Hypothesis for Transport Vesicle Targeting and Fusion,S
21、pecificity in vesicle docking and fusion is thought to be attained through specific interactions between specific v-SNARE proteins on the vesicle membranes and t-SNARE proteins on the membranes of the target compartment. SNAREs are a protein family. There are at least 20 different SNAREs in an anima
22、l cell.,The role of SNAREs in guiding vesicular transport.,The struction of paired SNAREs (the four-helix bundle). The SNAREs responsible for docking synaptic vesicles at the plasma membrane of nerve terminals consist of three proteins.,The basic molecular components in eukaryotic cells include v-SN
23、AREs (v-SNAP receptors) on transport vesicles, t-SNAREs (t-SNAP receptors) on target membranes, Rab GTPase, NSF, and several SNAPs. SNAPs are soluble NSF attachment proteins.,The role of Rab proteins in facilitating the docking of transport vesicles. GEF in donor membrane; GTP binding alters the con
24、formation of Rab, exposing its covalently attached lipid group and helps anchoring; Rab and Rab effectors help the vesicle dock and pairing of the v-and t-SNAREs; GDI: GDP dissociation inhibitor.,kinesin,dynein,C. Motor proteins mediate movement of vesicles along MT,A General Model for Kinesin-and Dynein-mediated Transport in a Typical Cell,D. Maintenance of membrane asymmetry,E. Membrane flow,作 业,膜泡表面标志与膜泡运输 过氧化物酶体中蛋白质的来源及输入机制,