1Growth Hormone & its Receptor (Growth Hormone & its Receptor (extracellular extracellular domains)domains)7.61 Eukaryotic CellBiology: Principles andPractice2004Lecture 8November 9, 2005Membrane Traffic7.61 Eukaryotic Cell7.61 Eukaryotic CellBiology: Principles andBiology: Principles andPracticePractice20042004Lecture 8Lecture 8November 9, 2005November 9, 2005Membrane 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?2Vesicles everywhere!Role in transportA schematic representation of four different models of intra-Golgi transport. a | In the vesicular transport model, coatomer protein complex-I(COPI) vesicles carry cargo and move in an anterograde fashion from one Golgi cisterna to the next. b | In the cisternal maturation model,the COPI vesicles move in a retrograde fashion and function as a retrieving device that is used by Golgi enzymes to maintain their specificand differential localization over the Golgi stack. ..c | The hybrid model proposes that COPI vesicles mediate both the anterogrademovement of cargo and the retrograde movement of Golgi-resident enzymes, and therefore possibly combines the vesicular transport andcisternal maturation models. d | The intercisternal connections model does not involve COPI vesicles and proposes that cargo and Golgi-resident enzymes move forwards and backwards, respectively, through tubules that connect the rims and the core of heterologouscisternae in a given Golgi stack.Schematic representation of four different models of intra-Golgi transport.Rabouille C, Klumperman J. Opinion: The maturing role of COPI vesicles in intra-Golgi transport. Nat Rev Mol Cell Biol. 2005 Sep 15;3Structure of the Golgihttp://www.rkm.com.au/CELL/organelles/organelleimages/golgi.jpg3D-Reconstruction fromEM serial sectionsGolgi Structure in Three Dimensions: Functional Insights from the Normal Rat Kidney 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 compartmentThe Golgi Ribbon:Stacks, vesicles, tubules4J 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!transcis5Marsh BJ. Lessons from tomographicstudies of the mammalian Golgi.Biochim Biophys Acta. 2005 Jul10;1744(3):273-92Golgi region of thepancreatic beta cell line,HIT-T15, visualized byhigh-resolution electrontomographyThe Golgi complex with the sevencisternae (cis–trans: C1–C7) is at thecenter. The color coding is as follows:C1, light blue; C2, pink; C3, cherry red;C4, green; C5, dark blue; C6, gold; andC7, bright red. The Golgi is displayed inthe context of all surroundingorganelles, vesicles, ribosomes, andmicrotubules: endoplasmic reticulum(ER), yellow; membrane-boundribosomes, blue; free ribosomes,orange; microtubules, bright green;dense core vesicles, bright blue;clathrin-negative vesicles, white;clathrin-positive compartments andvesicles, bright red; clathrin-negativecompartments and vesicles, purple;and mitochondria, dark green.Coated Vesicle BuddingUncoating of vesiclesSNARE-mediated Fusionto target membrane6SNARE-mediatedMembrane fusionDecision points:1. Cytosol: cytosol or organelle7Decision 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