Naren 5 and 6

Naren Lecture 5:
Morning vesicular trafficking secretion, afternoon endocytosis.
Will test on objective 4 from the Morning’s lecture.
See red box slide 4. We are in Golgi- proteins destined to go outside cell or to lysosome.
"Regulated" means you need a signal for vesicles to secrete out.
"Constitutive" means protein is secreted constantly.
Albumin is an example of a constitutively-secreted protein.
There are differences between constitutive protein and regulated protein.
Regulated ones come from a common pool of vesicles. Selective protein aggregated in electron- dense packing. Express unique proteases.
9
Vesicle buds- brings bound ligand into cell.
Recycles.
How do we study pathways?
10:
Label and follow thorugh pathways. They use a temperature sensitive protein. With a high temperature folding defect, the protein gets stuck in ER. If you reduce the temperature, the protein folds properly. By 180 min- protein goes to the to cell surface. Graph- compare how much is where. Add cycloheximide- suppresses further synthesis so you can view trafficking.
Proteins receive further sugar modification in Golgi.
How to discriminate sugars- SDS Page- if the protein has added sugars, molecular weight shifts upwards (slide 16). Form higher in gel is sensitive to endoglycosidase D. You can follow which proteins are in the sensitive form by how they migrate before and after digestion. Densitometric scan tells you how much protein is coming out.
18:
wild type yeast secrete normal protein. Mutant yeast- identify pathway from ER to Golgi (cis, medial, trans) to vesicles to surface.
19:
Classical experiment- N-acetylglucosamine transferase present in wild type. Without it other residues not added on.
Took medial Golgi from cells without VSV infection. G protein and infected mutant cells (without the NAG transferase) and mixed. Found addition of NAG to G protein.
21
Coat has 2 principal functions:
1. concentration of specific membrane proteins into patches to help select protein in the vesicle for transport.
2. Assembly of coat proteins into curved lattice defines vesicle of uniform size for its type
22
Coat proteins in green. Fusion- coat falls off and snare protein bring membranes close together to fuse.
24:
Kinds of coats:
COPI, COPII, Clathrin.
Concentrate on COPII.
25:
How do we know coat is formed?
Purified ER membrane and saw electron dense areas (26).
What is molecular switch (on test): Slide 27. SAR 1 is switch. Binds Sec12, GTP exchanges for GDP, Coat forms, Coat excised off by hydrolysis of GTP to GDP.
How does assembly take place?
SEC24 has binding to cargo protein (slide 28). Hydrolysis- N terminal tail withdraws from membrane..
How do we know GTP is required?
Add as on slide 29. GTP gammaS is a nonhydrolysable form of GTP. In presence of this substance, the coat accumulates and can’t come off.
30:
Know circled area.
Know SAR1, GTP exchange, Sec23 promotes hydrolysis of Sar1.
Transport can be Anterograde (Forward) and retrograde (Back).
Why is there retrograde transport?
Things can get lost and come back. If a protein gets mis-sorted, some receptors bind it and it goes to COPI pathway and comes back.
PDI binds KDEL receptor and signals to go back.
35:
How does fusion take place?
Vis vesicle snare, t is target snare.
Snare hypothesis- 1991- there are SNARES that bind to each other and lead to the fusion process. Has been revised.
37
fused SNARE complex is interacting in parallel 4-helix bundle. 2 from SNAP25, 1 from syntaxin , 1 from VAMPII. V snare and t snare were all that was required for fusion.
NSF and SNAP have ATPase activity and can disassemble the SNARE complex.
40 Snare well studied in neurotransmission.
42- synaptotagmin is Ca sensor.
Botox acts on snare protein. Botulinum toxins and tetanus toxins can act on proteins.These toxins have been shown to inhibit neurotransmission
Review: docking and fusion, Rab, assembly of SNARES, membrane fusion, disassembly.
How do membranes fuse- highly debated.
47
Hemagluttinin can bind to sialic acid at globular domain. In endosome, pH is low. Conformational change and globular domain falls off as helical domain enters membrane.
It is like a spring-loaded gun.Membranes come close together and open up. SNARES may act like this, too.
50.
Summary
Review Role of SAR1 in assembly and disassembly of COPII coats. 5 steps.1. GTP exchange
2.Coat assembly
3. cargo protein
4 GTP hydrolysis
5. coat disassembly. Then review docking fusion, SNAREs.
He will post review slides.
Lecture 6-Thank You to Daniel!!!
Danielnotes—"The backup’s backup!"TM
Cell and Molecular Biology
A.P. Naren Ph. D
Lecture 6: Vesicular Traffic, Secretion, and Endocytosis
Note: the order he went in was different that the order the slides are arranged in the handout. Numbers in parentheses (ie. (6)) indicate the slide number from the PowerPoint that’s on Blackboard. I only reproduce here what was not on the slides themselves. A slide without any comment indicates that Dr. Naren added nothing that is not on the slide.
BOLD headings mean the slide is on the examination
(1) Title. Endocytosis is the internalizing of the outside of the cell. We will also talk about lysosomal traffic today.
(8) Objectives: only objective 4, receptor mediated endocytosis, is on the test
(2) Both exocytosis and endocytosis happen together as a constant process. Exocytosis secretes cargo out. Endocytosis internalizes cargo-containing vescicles.
(10)
(11) Clathrin is a protein that is a major constituent of vesicles in endocytosis
(9) Clathrin forms a cage around vesicles
(12) Clathrin forms the Triskelion structure
(13) The sizes of each chain are on the slide and were emphasized.
(14) 10 Triskelion form the assembly intermediate
(15) A movie on clathrin assembly. On Blackboard, perhaps?
(17) Dynamin, a GTPase, performs a pinching action, pinching off the vesicles
(18) Dynamin acts as a noose, pinching off the vesicle in response to GTP hydrolysis
(19) GTP hydrolysis is critical for this process
(20) In the flies in this example, there is a dynamin mutation. Protein is stuck in the membrane at 30 degrees C. At 20 degrees C, the protein folds normally, and escapes the membrane without incident
(Not in power point) What determines lysosome targeting?
(22) Manose 6 phosphate determines lysosome targeting via a two-step reaction process
(23)
(24)
(25) He spoke about Hurler’s syndrome and I-cell disease, the difference between the two being the lack of M6P in I-cell disease.
(26) A sugar enzyme is missing in Fabry’s disease. It also leads to mental retardation and death
(27)
(28) Transcytosis—both endocytosis and exocytosis in combination. In this diagram, the apical (luminal) side is on the right, and the basal (blood) side, on the left
(29) A good example of transcytosis is the transmission of maternal IgG antibodies from ingested breast milk to the neonatal blood stream via intestinal lumen absorption
(30)
(31)
(32) This topic is ON TEST. Receptor mediated endocytosis—the budding of vesicles from the plasma membrane, bringing specific bound ligands into the cell
(33) LDL particles hit LDL receptors in an LDL binding domain. It binds at pH 7 with high affinity.
(34) NPXY is the sorting signal (the X meaning any amino acid) and corresponds to YXXphi. The sorting signal binds to the AP2 complex. Then clathrin is recognized and the cage is formed
(35) LDL-ferritin here is electron dense
(36) LDL-ferritin here is inside the vesicle
(37) Don’t forget that dynamin is needed for this process
(39) Especially noted was the step where the clathrin coat is shedded
(38) When the vesicle fuses to the endosome, the endosome has a lower pH than the lumen
(41) All this occurs at pH 7.0
(42) All this occurs at the lower pH of the endosome, pH 5.0. The LDL particle is released
(40) Dark spots here have been labeled with gold
(39) After delivering the LDL to the endosome, the receptor is returned to the cell surface via another vesicle. The LDL goes into the lysosome for recycling and use.
(Not in powerpoint) Endocytosis can regulate the number of G-protein cell receptors at the cell surface
(44) When activated, the G-protein receptor is phosphorylation and other bits bind to it. These bits pull it into the cell to desensitize the surface, thus preventing hypersensitivity
(45) Number 2, autophagic enzymes digest bits of the cell itself in starvation type situations
(46) Only proteins that are slightly deranged are pulled in via the autophagic mechanism
(47) For example, viruses are internalized. The key here is the replicated viruses that bud outward
(48) ESCRT complex—ubiquitin binds to it. It is ATP dependent. It either internalizes cargo via multivesicles, or causes budding of cargo-containing vesicles
(49) (b) here is a case with a mutant ESCRT, so no budding can occur
(50) Important to remember from here are:
Clathrin, Adaptors, and dynamin in endocytosis
Sorting signal (YXXphi)
Dissociation (importance of pH)
(53) A schematic overview of the receptor mediated endocytosis pathway. Note Sorting signal, dissociation, and lysosome here.
(54)
At this point, Dr. Naren opened a new slideshow for exam review. It mainly consisted of slide 51 from our powerpoint, with a few alterations, which served to concisely summarize all his lectures. The slide he showed read the following :
REVIEW1. The importance of ER in protein translocation
What is cotranslational and post-translational translocation?
How are proteins inserted into ER membranes?
2. Glycosylation, disulfide bond formation
- Proper folding, and degradation of misfolded protein
3. Nuclear transport: Import (NLS signal) and export
4. Mitochondrial and peroxisomal protein transport
5. Vesicular traffic, secretion/exocytosis: Sar1 and COPII, docking and fusion of vesicles
6. Vesicular traffic, Secretion and Endocytosis: Receptor mediated endocytosis
Dr. Naren then gave a brief overview of each lecture that he had given. This provided no new information than that which he already gave in previous lectures, save that, for point 3, the RAN cycle and exchange factor are important.
Daniel Taylor
11/19/2006