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1、Protein Sorting,Chapter 6 II,Overview of sorting of nuclear-encoded proteins in eukaryotic cells,Proteins are imported into organelles by three mechanisms: Gated Transport: Transport through nuclear pores Transmembrane transport: ER, Mit, Chl, Per Vesicular transport: ER-Golgi-PM-Lys, Endosome,Road
2、map of protein sorting,Protein sorting: Protein molecules move from the cytosol to their target organelles or cell surface directed by the sorting signals in the proteins.,Signal peptides and Signal patches,Figure 6-8 Two ways that a sorting signal can be built into a protein. (A) The signal resides
3、 in a single discrete stretch of amino acid sequence, called a signal peptide, that is exposed in the folded protein. Signal peptides often occur at the end of the polypeptide chain, but they can also be located elsewhere. (B) A signal patch can be formed by the juxtaposition of amino acids from reg
4、ions that are physically separated before the protein folds; alternatively, separate patches on the surface of the folded protein that are spaced a fixed distance apart could form the signal.,Gated transport:,Through gated poresNuclear pores; Nuclear localization signal (NLS); Folded and assembly fo
5、rm to transport.,Transmembrane transport,ER signal sequence, Mit, Chl, Per: Leader sequence; Through translocation on the membrane; Single and Unfold form; Helped by molecular chaperons,Vesicular transport,Budding, transporting, docking and at last fusion with target membrane; Assembly coated protei
6、ns on the vesicles (Clathrin, COPII and COPI); Only Properly folded and assembled proteins; The orientation of transported proteins and lipids is not changed during transporting.,B. Signal Hypothesis A model for the Signal Mechanism of Cotranslational Import -G.Blobel & D.Sabatini,1971.,Evidence Tha
7、t Protein Synthesized on Ribosomes Attached to ER Membranes Pass Directly into the ER Lumen Milstein : IgG,Milstein et al: Studying the synthesis of light chain of IgG (in cell-free systems, 20 Aa longer at N-terminal end than the authentic light chain ),Adding ER membranes to this system leads to t
8、he 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,Signal-recognition particle, SRP: Six different polypeptides complexed with a 300-nucleotide (7S)molecule of RNA. ER signal sequence: Typic
9、ally 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 cleavage from the mature protein will take place. A signal sequence on nascent seretory proteins targets them to the ER and is then clea
10、ved 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 to block further translation; One that binds to the ER membrane (docking protein),The sorting signal of VSV glycoproteins : Asp-X-Gln
11、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 simultaneously with VSV and influenza virus, the VSV glycoprotein is found only on the basolateral membrane, whereas the HA glycoprotein of th
12、e 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 basolateral membrane via specific transport vesicles that bud from the trans-Golgi network. In certain other polarized cells, some ap
13、ical and basolateral proteins are transported together to the basolateral surface; the apical proteins then move selectively, by endocytosis and transcytosis, to the apical membrane. K. Simons et al., Cell 62:207; K. Mostov et al., JCB. 116:577,Start-transfer Sequence & Stop-transfer Sequence,Figure
14、 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 a helix functions as an internal, uncleaved signal-anchor sequence (red), directing binding of the nascent polypeptide chain to the r
15、ER 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 stop-transfer membrane-anchor sequence, extrusion of the chain through the translocon into the ER lumen ceases. The first two a helices
16、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 cytosol. Subsequent a-helical hairpins could insert similarly, although SRP and the SRP receptor are required only for insertion of t
17、he first signalanchor sequence. Although only six transmembrane a helices are depicted here, GLUT1 and proteins of similar structure have twelve or more. H. P. Wessels et al., Cell 55:61.,The Orientation of Nascent Polypeptide,The Nascent polypeptide is oriented within translocon so that the positiv
18、ely charged flanking sequence faces the cytosol,C. A Model for the Postranslational Import of Polypeptides into the Mit.,Post-translational modification and quality control in the rER,Disulfide bonds are formed and rearranged in the ER lumen Only in ER lumen is there a redox environment for oxidatio
19、n 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,Proteins without any signal sequence are cytosol residual proteins,D.Types of Vesicle Transport and Their Functions,(A). The three differe
20、nt types of coated vesicles. Different coat proteins select different cargo and shape the transport vesicles that mediate the various steps in the biosynthetic-secretory and endocytic pathways.,COPII-coated vesicles move materials from the ER to the Golgi.,The assembly of a COPII-coated vesicles. Sa
21、rGTP 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 transport from one Golgi compartment to another in mammalian cell.,COPI-coated vasicles transporting Escaped ER resident Proteins Ba
22、ck 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. COPI coated vesicles may select specific cargo. ER is an open prison. Soluble ER protein bear Retrieving signalKDEL(Lys-Asp-Glu-Leu)in
23、 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-coated vesicle were first identified by treatment of GTP analogues - COPI-coated vesicle accumulated within the cell and could be iso
24、lated 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 transport vesicles back to the ER. Neutral pH: dissociate from the KDEL; low pH: binding the KDEL,Clathrin-coated vesicle: Transport
25、ing 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 of Golgi is the Sourse of Clathrin-coated vesicle. (2) Clathrin-coats contain: protein clathrin-which forms a structural scaffold, a
26、daptors- 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,Specificity in vesicle docking and fusion is thought to be attained through specific interactions between specific v-SNARE proteins
27、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 animal cell.,The role of SNAREs in guiding vesicular transport.,The struction of paired SNAREs (the four-helix bundle). The SNAREs respo
28、nsible for docking synaptic vesicles at the plasma membrane of nerve terminals consist of three proteins.,The basic molecular components in eukaryotic cells include v-SNAREs (v-SNAP receptors) on transport vesicles, t-SNAREs (t-SNAP receptors) on target membranes, Rab GTPase, NSF, and several SNAPs.
29、 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 conformation 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,