*note: The format of this section will be a lecture on background material on April 21 st then discussion of papers on April 26 th
and 28 th
. There will be a take home exam given out on April 28 th
, due May 3 rd
. My goal for this section of the course is for you to not only understand the endocytic and exocytic pathways, but also for you to understand the techniques that are used to study these processes. For example, how were all of those protein components involved in the secretory pathway listed in the book originally identified and characterized? How do you know that a protein is endocytosed? How do you know it recycles to the membrane?

Relevant sections from book, as listed.

2-3 research papers per day. Focus on the experiments. Be prepared to discuss the figures.
It is expected that these assignments will be your own work. Prior to class you may compare notes, and can make corrections, but you must read and analyze the papers on your own. You may not use notes from students who have taken this class or BCH 705 in previous years.
 Summary of research papers (due on day papers are discussed)

Overview : 2 sentences- what were they trying to do?

Materials and Methods : Brief outline of experimental system.

Results :
For each figure or table, discuss briefly:
1) Purpose of experiment.
2) Briefly , what was done.
3) Controls done, and any controls missing.
4) Results and conclusions.

Discussion : What did they learn?

TAKE HOME EXAM (DUE MAY 3 rd
, at start of class) You may use your book, your own notes, and the papers to answer these questions. Make sure your notes are complete before April 28 th
. After the exam is handed out, you may not get help from any other person, including getting their notes.
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Lecture on the reading from the textbook (see below) and background material for the papers.
Three papers on the molecular mechanisms of exocytosis. The fourth paper is for your reference. The first two papers illustrate the basic techniques that were used to determine the role of all of the proteins involved in membrane trafficking described in your book. They are taken from a book published by the
American Society for Cell Biology "Landmark Papers in Cell Biology". The third paper describes experiments to settle a dispute that arose in the field based on differences seen using the genetic approach (Schekman) vs. the biochemical approach (Rothman). Try and figure out what that controversy is. It is also one of the cleverest sets of experiments I know of.
1) Novick, Field, and Scheckman, 1980, Cell, Vol. 21, pp. 205-215.
2) Balch, Dunphry, Braell, and Rothman, 1984, Cell, Vol. 39, pp. 405-416.
3) Hu, Ahmed, Melia, Sollner, Mayer, Rothman, 2003, Science Vol. 300, pp.1745-9.
Background: Cook and Davidson, 2001, Traffic, Vol. 2, pp. 19-25
Three papers on the membrane trafficking of the insulin-responsive glucose transporter GLUT4 and its companion protein IRAP/VP165. These proteins cycle from intracellular compartments to the plasma membrane in response to insulin (regulated exocytosis) and are recycled back to the internal pool for reuse (endocytosis). See below for background information. I chose these papers because they use a wide variety of tools that are commonly used to study endocytic membrane trafficking, such as subcellular fractionation and internalization of surface labeled proteins. These techniques and what they tell us about the pathway utilized by GLUT4 will be the focus of our paper discussions.
1)
2)
3)
Cain, Trimble, and Lienhard, 1992, J. Biol. Chem. Vol. 267, pp. 11681-11684.
Hashiramoto and James, 2000, Mol. Cell. Biol. Vol. 20, pp. 416-427.
Garza and Birnbaum, 2000, J. Biol. Chem. Vol. 275, pp. 2560-2597.
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st
Chapter 12, Intracellular compartments and protein sorting p. 659-662- Organelles p. 666-667- Signal sequences p. 668- Approaches to study signal sequences. p. 689-702- Protein sorting and translocation into the endoplasmic reticulum p. 702-703- Glycosylation
Topography of membrane proteins-
Type I proteins- amino terminus in vesicle lumen/outside of cell, carboxy terminus in cytosol. Examples: insulin receptor.
Type II proteins- amino terminus in cytosol, carboxy terminus in vesicle lumen/outside of cell. Examples: transferrin receptor, IRAP (insulin-responsive aminopeptidase;
VP165).
Multipass transmembrane proteins- multiple membrane spanning domains with alternating cytosolic and luminal/extracellular loops. Examples: glucose transporters
(GLUT4, GLUT1) have 12 transmembrane domains, both amino-terminus and carboxyterminus cytosolic, with 5 intracellular and 6 extracellular connecting loops.
Chapter 13, Intracellular Vesicular Traffic p. 711-726- General principles and molecular mechanisms
Clathrin, COP I and COPII coated vesicles; NSF and

/

/

SNAP; v-SNARE/t-
SNARE/SNAP 25 complexes; Rab proteins, ARF's, and other small GTP-binding proteins.
ER to Golgi to post-Golgi sorting compartment (trans-Golgi reticulum, TGN) to cell surface or lysosomes; vesicular transport vs. "cisternal progression"; integral membrane vs. secreted proteins. p. 714-715- Approaches to study vesicular transport p. 726-727- ER to Golgi transport, COPII vesicles p. 729-730- KDEL sequence and membrane/protein retrieval. Retrograde transport
from the Golgi to the ER
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p. 731-734, 736-737- Golgi and oligosaccharide processing. The Golgi is an ordered
series of compartments.
N-linked oligosaccharides- addition of high mannose GlcNAc and initial processing in
ER. Trimming of mannose and addition of GlcNAc, fucose, galactose, and sialic acid in the cis , medial , and trans Golgi Note that different enzymes and processes occur in separate stacks of the Golgi (enzyme composition as well as position defines each cisternal compartment). Sequential processing occur in sequential cisternae. p. 739-740- Lysosomes p. 742-743- Mannose-6-phosphate signals and receptors and transport to lysosomes. p. 746-753- Endocytosis
Fates of endocytosed proteins:
1) recycled for reuse- transferrin, transferrin receptor, insulin receptor, LDL
receptor.
2) delivery to lysosomes for degradation- LDL, insulin. p. 757-761- Exocytosis: regulated vs. constitutive secretion
Note: The endocytic recycling compartment is what I refer to as the "early endosome", and a second compartment that contains the mannose-6-phosphate receptor and newly synthesized hydrolytic enzymes from the Golgi is what I refer to as the "late endosome". Material is endocytosed and delivered first to the early, recycling endosomes, then the portion that is not recycled becomes the late endosome, where newly synthesized lysosomal enzymes are delivered. Material is transported from the late endosome to the mature lysosomes via small vesicles.
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The experiments described in the assigned papers focus on identification of the endocytic compartments involved in GLUT4 trafficking. An understanding of this pathway may lead to insights into the molecular mechanism by which insulin stimulates glucose transport, and perhaps to novel targets for the treatment of diseases in which this process is abnormal, such as in type II non-insulin dependent diabetes mellitus.
In the peripheral, insulin-responsive tissues (muscle and adipose tissue), glucose transport is tightly regulated. Under basal conditions, glucose transport into these tissues is low, but after insulin stimulation the rate of glucose uptake increases dramatically, up to 50-fold in isolated adipocytes.
Exercise also stimulates glucose transport in muscle. The glucose transport in these insulin-responsive tissues is regulated through the insulin-responsive isoform of the glucose transporter, GLUT4. All tissues express a closely related, constitutive glucose transporter, GLUT1. In primary adipocytes, the levels of expression of GLUT1 are quite low, but in the 3T3-L1 adipocyte tissue-culture model, GLUT1 levels are elevated.
The glucose transporters are facilitative transporters. They specifically transport glucose across the membrane, but can do so bi-directionally. Glucose is transported down a concentration gradient into the cell, where it is immediately converted to glucose-6-phosphate by hexokinase. This keeps the intracellular concentration of free glucose very low. Since the GLUTs cannot transport glucose-6-phosphate, glucose transport is uni-directional into the cell. Although it is somewhat controversial, it does not appear that the intrinsic activity of GLUT4 can be regulated. Therefore, the GLUT4 in the membrane can be thought of as a channel that allows glucose to pass across membranes.
The mechanism of regulation of glucose transport in adipocytes and muscle involves regulation of the total number of GLUT4 glucose transporters expressed at the cell surface. Under basal conditions
GLUT4 is found only at very low levels at the plasma membrane (less than 1% of the total cellular
GLUT4 is found at the plasma membrane, PM, under basal conditions). The GLUT4 in the cell is localized to small tubular-vesicular compartments found throughout the cytoplasm and in the trans-golgi region of the cell (low density membrane fractions, LDM; the high density membrane fractions, HDM, contain both intracellular vesicles and plasma membrane-derived vesicles). In response to insulin, these vesicles move to and fuse with the plasma membrane, resulting in a 50-fold increase in GLUT4 at the plasma membrane in adipocytes (after insulin stimulation, 50% of the GLUT4 is found at the plasma membrane). This is not a static delivery process, however. The GLUT4 at the plasma membrane is continuously internalized and recycled via the clathrin-coated pits and the endocytic pathway under both basal and insulin-stimulated conditions. The primary effect of insulin is to stimulate the rate of exocytosis of GLUT4. The rate of endocytosis of GLUT4 also decreases (to a lesser extent), leading to an overall increase in the ratio of external GLUT4 to internal GLUT4. When insulin is removed, the rates of exocytosis and endocytosis of GLUT4 return to basal levels and the GLUT4 redistributes to its intracellular localization. A similar pathway is seen for the recycling of the membrane components of synaptic vesicles and regulated secretory vesicles.
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Some additional information:
IRAP/VP165/GP165- insulin-responsive amino-peptidase: This is a 165 kD glycoprotein that is a type II single-transmembrane domain membrane protein. It was originally identified by Kostia
Kandror and myself, working as post-docs in two separate labs, as a protein component of
GLUT4-containing compartments. This protein colocalizes with GLUT4 completely and translocates with GLUT4 to the plasma membrane with insulin-stimulation, in a manner identical to GLUT4. It is used as a second marker of the GLUT4 compartment, and is used in the third paper instead of GLUT4 because 1) GLUT4 is very inefficiently labeled at the cell surface, and 2)
IRAP has only a single transmembrane and intracellular domain, unlike the 7 domains in GLUT4.
(IRAP has an identical topography to the transferrin receptor.) Therefore, it is expected that sorting signals that account for the highly specialized membrane trafficking of IRAP (and
GLUT4) will be more easily determined in IRAP than in GLUT4.
TfR- transferrin receptor; also transferrin: These proteins are only internalized as far as the early, sorting endosome and are markers of this compartment. As described in the book, they are involved in the uptake and delivery of iron in all cells.
Amine oxidase and sortilin: Two proteins that were also found in GLUT4 vesicles.
CIMP/IGF-II receptor: cation independent mannose-6-phosphate receptor/insulin-like growth factor receptor- cell surface receptor that internalizes to the early endosome.
CDMPR- cation-dependent mannose-6-phosphosphate receptor: the receptor in the book that cycles between the golgi and the late endosome (some also goes to the PM) that delivers newly synthesized lysosomal enzymes.
Rab4/11: small GTP binding proteins involved in membrane trafficking (many others known) each compartment has a particular assortment of Rabs.
Cellubrevin/VAMP2: isoforms of synatobrevin involved in membrane trafficking, v-SNARES.
Syntaxin 6 (t-SNARE) and asiologlycoprotein: Trans-Golgi network markers.
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