1Growth Hormone & its Receptor (Growth Hormone & its Receptor (extracellular extracellular domains)domains)7.61 Eukaryotic CellBiology: Principles andPractice2004Lecture 82005Membrane Traffic7.61 Eukaryotic Cell7.61 Eukaryotic CellBiology: Principles andBiology: Principles andPracticePractice20042004Lecture 8Lecture 820052005Membrane TrafficMembrane TrafficSecretory Pathway and Membrane TrafficQuestions arrising:0. How do you break the membranebarrier?1. What happens to a protein as ittravels thru the pathway?2. Why so many differentcompartments? How are theymaintained in spite ofintercompartmental flow3. What are the signals on theprotein?4. What cellular machinary isinvolved in decoding the signals?5. How is specificity generated(specificity of cargo, specificity oftargeting)?6. How do you go about studyingsuch complex pathways?Vesicles everywhere!2Golgi Structure in Three Dimensions: Functional Insights from the Normal RatKidney CellMark S. Ladinsky, David N. Mastronarde, J. Richard McIntosh, Kathryn E. Howell, and L. Andrew StaehelinJ. Cell Biol., Volume 144, Number 6, March 22, 1999 1135-1149ERGIC - ER/Golgi intermediate compartmentJ Cell Biol. 1999 Mar 22;144(6):1135-49. Related Articles, Links Click here to read Golgi structure in three dimensions: functional insights from the normal rat kidney cell. Ladinsky MS, Mastronarde DN, McIntosh JR, Howell KE, Staehelin LA. Laboratory for Three-Dimensional Fine Structure, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347, USA. Three-dimensional reconstructions of portions of the Golgi complex from cryofixed , freeze-substituted normal rat kidney cells have been made by dual-axis, high-voltage EM tomography at approximately 7-nm resolution. The reconstruction shown here ( approximately 1 x 1 x 4 microm3) contains two stacks of seven cisternae separated by a noncompact region across which bridges connect some cisternae at equivalent levels, but none at nonequivalent levels. The rest of the noncompact region is filled with both vesicles and polymorphic membranous elements. All cisternae are fenestrated and display coated buds. They all have about the same surface area, but they differ in volume by as much as 50%. The trans-most cisterna produces exclusively clathrin-coated buds, whereas the others display only nonclathrin coated buds. This finding challenges traditional views of where sorting occurs within the Golgi complex. Tubules with budding profiles extend from the margins of both cis and trans cisternae. They pass beyond neighboring cisternae, suggesting that these tubules contribute to traffic to and/or from the Golgi. Vesicle-filled "wells" open to both the cis and lateral sides of the stacks. The stacks of cisternae are positioned between two types of ER, cis and trans. The cis ER lies adjacent to the ER-Golgi intermediate compartment, which consists of discrete polymorphic membranous elements layered in front of the cis-most Golgi cisterna. The extensive trans ER forms close contacts with the two trans-most cisternae; this apposition may permit direct transfer of lipids between ER and Golgi membranes. Within 0.2 microm of the cisternae studied, there are 394 vesicles (8 clathrin coated, 190 nonclathrin coated, and 196 noncoated ), indicating considerable vesicular traffic in this Golgi region. Our data place structural constraints on models of trafficking to, through, and from the Golgi complex.250 nm 3D EM reconstruction of the 3D EM reconstruction of the GolgiGolgi 3D EM reconstruction of the 3D EM reconstruction of the Golgi Golgi ::vesicles (white), vesicles (white), ciscis (light blue), (light blue), transtrans (orange & red), (orange & red), medialmedial (other) (other)Vesicles everywhere!transcis3Coated Vesicle BuddingUncoating of vesiclesSNARE-mediated Fusionto target membraneSNARE-mediatedMembrane fusionDecision points:1. Cytosol: cytosol or organelle4Decision points:1. Cytosol: cytosol or Organelle (focus on ER,also mitochondrion and peroxisome andnucleus)2. ER lumen: modifications, proper folding andtransfer to Golgi (conformation, assembly, Bip)3. transitional ER or cis golgi: return to ER(KDEL sequence)4. cis golgi: modify for lysosomal targeting5. cis golgi: transfer to medial golgi6. medial golgi: modifications and transfer totrans golgi7. trans golgi: modifications and transfer to TGN8. TGN: modifications, constitutive or regulatedsecretion9. TGN: lysosomal or cell surface targeting (orretrograde)10. TGN: differential sorting to polar surfaces(apical, basolateral)11. surface: stable expression/recycling12. endosomes: return to surface, transcytosis,return to Golgi, degradationThe apical surface is red,basal and lateral portions of the basolateral surface are green and blue, respectively.Known components of the trafficking machinery specific to each pathway are indicated. AEE, apical early endosome ;ARE, apical recycling endosome; BEE, basolateral early endosome ; CE, common endosome ; LE, late endosome.Polarized epithelial membrane traffic: conservation and plasticity.Mostov K, Su T, ter Beest M.Department of Anatomy, Genentech Hall, 600 16th Street, University of California,San Francisco, CA 94143-2140, USA. [email protected] cells are polarized and have distinct plasma membrane domains, which are theresult of polarized trafficking of proteins and lipids. Great progress has been made inelucidating the highly conserved polarized targeting machinery. A pre-eminentchallenge now is to understand the plasticity of polarized traffic, how it is altered bydifferentiation and dedifferentiation during development, as well as the adaptation ofdifferentiated cells to meet changing physiological needs.Polarized epithelial membrane traffic: conservation and plasticity. Mostov K, Su T, ter Beest M. Nat Cell Biol. 2003 5(4):287-93.Polarized epithelial membrane traffic:Biosynthetic Apical and BasolateralSortingLipid rafts,Glycosylation?AEE5Signal recognition particle (SRP) mediatedtranslocation of proteins into the ER (translationalarrest, SRP receptor, sec61 pore, signal peptidase,etc. - assume you know this!)Cotranslational (ER) and posttranslational (ER,Golgi, TGN) processing of newly synthesizedproteinsSelected topics in secretion:A. glycosylation: N-linked (Asn) ,O-linked (Ser,Thr) andproteoglycans (Ser,Thr) (disacharides (uronicacid+GalNAc or GlcNAc), O-GlcNAcB. fatty acylation & polyisoprenylation: palmitate(myristate on