Review for MT2

Question 1

Identification of SNAP receptor.

Answer 1

1. Purify myc-tagged NSF made in E. coli and couple to anti-myc agarose beads
2. Add purified SNAPs
3. Add detergent extracted membranes (should contain SNAREs the putative integral membrane protein)
4. Wash
5. Purify antibody beads and all associated proteins
6. Elute
7. Run out on SDS-PAGE
8. Excise band and sequence

Question 2

Modified SNARE hypothesis and Rab tethering.

Answer 2

• Specific Rabs on both the vesicle and target membrane – Rab-GTP associates with membranes via a hydrophobic domain region that inserts into the membrane
o Localized GEFs contribute to Rab localization
• Rab effector (tethering protein) is associated with the target membrane and Rab GTPase
• Rab-GTP on vesicle binds to tethering protein on the target
• Tethering brings membranes close enough to allow SNARE interaction
• v-SNAREs on the vesicle directly associate with t-SNARES on the target membrane to facilitate docking and fusion (one v-SNAREs are integral membrane proteins with a-helical structure and t-SNAREs (2 or three of them), at least one is an integral membrane protein; others may be peripheral, similar a-helical structure)
• Fusion of vesicle and target membrane leads to release of contents and the formation of the trans-SNARE complex, whatever was in the vesicle membrane is now in the target membrane
• SNAPs bind to trans-SNARE complex and recruit NSF which, in the presence of ATP, mediates SNARE dissociation through ATP hydrolysis activity

Question 3

Transport from ER to Golgi using the bulk transport pathway.

Answer 3

1. Proteins folded by calnexin are either ER resident or need to leave the ER. The latter proteins have exit signals.
2. Cargo receptors, that also have exit signals, bind to cargo
3. Sometimes, ER resident proteins also get taken up by the bulk pathway
4. A COP2 coated vesicle buds off from the ER, gets uncoated and comes off the ER, Sar1 is the coat recruitment GTPase
5. Two identical Cop II vesicles undergo homotypic fusion
   a. NSF pries apart trans-SNARE complex
   b. SNAREs interact between vesicles to mediate fusion to form vesicular tubular clusters
   c. Rab proteins also mediate this step
6. Vesicular tubular clusters are moved along by motor proteins associated to microtubules and evolve into the cis Golgi

Question 4

Golgi (or vesicular tubular cluster) to ER retrieval pathway

Answer 4

1. ER resident proteins have KDEL sequences and bind to KDEL receptor in more acidic environment of the Golgi (relative to the ER)
2. KDEL receptors and their KDEL cargo are taken up in COPI coated vesicles where ARf1 is the coat-recruitment GTPase
3. The vesicle fuses with the ER and releases its contents, the higher pH of the ER allows unloading of the ER resident protein
4. The KDEL receptor has an exit signal and interacts with COP II coated vesicles to return to the Golgi

Question 5

Constitutive secretory pathway.

Answer 5

1. Protein destined for release or to be membrane associated (not bound for the lysosome or in secretory vesicles) go through the non-selective default pathway
2. Proteins first cycle from stacks of the Golgi through COP I coated vesicles, which have the Arf1 GTPase
3. Vesicles containing the aforementioned proteins bud off in COP1-coated vesicles
4. Vesicles then fuse with the early endosome and then go to the cell membrane

Question 6

Regulated secretory pathway.

Answer 6

1. Specific proteins meant for secretion by specialized cells bud off of the TGN into secretory vesicles (vesicles bud off of TGN containing aggregates of secreting protein) (concentration mechanism unclear)
2. Vesicles accumulate near the cell surface
3. Hormone or other signal is required to allow vesicle fusion with cell membrane
   a. Signal often regulates Rab function

Question 7

Lysosomal acid hydrolase pathway to lysosome.

Answer 7

1. Properly folded lysosome resident protein has its signal patch exposed
2. GlcNAc phosphotransferase binds the protein via its signal patch at the recognition site in the cis Golgi and binds a UDP-GlcNAc at its catalytic site
3. GlcNAC phosphotransferase catalyzes the transfer of GlcNAC-P to Mannose on the lysosomal hydrolase
4. In the trans Golgi, a second enzyme cleaves the GlcNac, exposing the M6P signal
5. The higher pH in the TGN allows the M6PR to bind to the lysosomal hydrolase via its M6P N-linked oligosaccharide
6. These are then packaged into clathrin coated pits that bud off as clathrin coated vesicles
7. The uncoated vesicles then fuse with a late endosome and release their contents
8. The acid hydrolase is unloaded due to the lower pH of the late endosome
9. The acid hydrolase will then go to the lysosome whilst the M6PR will be recycled back to the Golgi via a retromer coated vesicle

Question 8

Neuron vesicle transport and glutamate vesicle recycling

Answer 8

1. NTs from the ER travel to the Golgi via COP2 coated vesicles
2. NTs travel through the Golgi via COPI coated vesicles
3. Secretory vesicles containing NTs bud off the TGN and go through the regulated secretory pathway to empty their contents for synaptic signaling
4. Vesicles are taken back and recycled by endocytosis
5. V-type ATPase pumps H+ into recycled vesicles to make the environment acidic
6. NT and H+ go through antiporter (Secondary active transport) to load NT into synaptic vesicles

Question 9

Receptor-mediated endocytosis for LDL

Answer 9

1. Cholesterol is transported in the blood as LDL (protein-lipid complex)
2. LDL binds LDL receptor on the cell surface
3. LDL binding triggers ubiquitination of the receptor cytosolic tail allowing interaction with clathrin adaptor proteins
4. LDL receptor and associated LDL are internalized via clathrin-coated vesicles
5. Vesicles fuse with the early endosome
6. Low pH of the early endosome causes separation of LDL from its receptor
7. Receptor is recycled back to the cell surface and the LDL gets transferred to the lysosome
8. Cholesterol eventually gets out of the lysosome and is transported to the ER to get incorporated into membranes

Question 10

Familial hypercholesterolemia

Answer 10

1. Cholesterol is transported in the blood as LDL
2. LDL binds to the LDL receptor
3. LDL receptor lacks part of the cytosolic tail that is normally mono-ubiquitinated
4. LDL receptor cannot interact with the clathrin adaptor proteins
5. LDL receptor cannot be internalized
6. Cholesterol accumulates in the blood

Question 11

Receptor mediated endocytosis for EGF

Answer 11

1. EGF binds to EGF receptor (interaction via EGF repeats) triggering clathrin-mediated endocytosis
2. Vesicle fuses with early endosome
3. Mono-Ubiquitin (x2) is covalently attached to the cytosolic tail of the EGFR and acts as a signal for internalization of the receptor
4. ESCRT proteins bind to endosomal PIPs and ubiquitin on the EGFR cytosolic tail
5. ESCRTs functions to cluster receptors and bring them to ESCRT-III which mediates invagination, forming a membrane bound vesicle inside the endosome
6. Early endosomes fuse to form multivesicular bodies that migrate along microtubules
7. The multivesicular body fuses with a late endosome or a lysosome
8. The EGF-EGFR complex are degraded in the lysosome, downregulating the signal

Question 12

Sevenless pathway

Answer 12

1. Boss transmembrane ligand binds to Sevenless RTK
2. Binding leads to homo-dimerization
3. Dimerization results in trans-phosphorylation of multiple sites leading to simulation of the higher kinase activity leading to compete activation
4. Drk SH2 domain protein binds to phosphorylated tyrosine on activated RTK
5. Drk recruits the Ras-GEG: Sos
6. Sos promotes the GDP to GTP exchange of membrane associated Ras, thus making it active
7. Ras activates Raf
8. Raf phosphorylates and activates Mek
9. Mek phosphorylates and activates MAPK
10. Activated MAPK enters the nucleus and phosphorylates transcription factors
11. Transcription factors promote transcription of genes required for R7 cell fate

Question 13

Mutagenesis screen to identify boss and sevenless.

Answer 13

1. Make millions of mutant flies and add them to chambers that have UV light shining at one end
2. Take flies that do not migrate towards the UV light (that isn’t due to inability to walk or fly)
3. Identify Boss and Sev

Question 14

Clonal analysis

Answer 14

1. Start with an individual in which all cells are heterozygous for boss and chp marker mutation (both are on the same chromosomes)
   a. These will be phenotypically wt since both mutations are recessive
2. Induce mitotic recombination
3. Obtain from a single cell either a homozygous wt cell or a cell that is homozygous for the mutations
4. These cells divide and make up a random assortment of cells that are either wt or mutant
5. Identify cells through chp marker
6. Boss needed in R8 cell, sev needed in R7 cell for R7 fate

Question 15

Identification of dominant enhancers of sev hypomorph.

Answer 15

1. Start with Sev-B4 temperature sensitive allele that is a sev hypomorph
2. Grow flies at 23C, where you still get R7 photoreceptors in all ommatidia (just barely however); cusp of functioning = just enough sevB4 to make pathway functional
3. Take SevB4 mutants and create a random mutation; large scale
4. Cross the mutation induced sevB4 flies with flies that are mutant for sevB4
5. Get flies that are homozygous mutant for sevB4 and heterozygous for the mutation
6. If the phenotype is wt (all ommatidia have R7) then the mutation has no effect
7. If the phenotype is lack of R7, the heterozygous mutation is a dominant enhancer of the sev hypomorph

Question 16

GST pulldown - drk and sos

Answer 16

1. Make a GST-Drk fusion protein in bacteria
2. Purify the recombinant fusion protein by binding to glutathione beads
3. Incubate the purified protein with cell lysates from bacterial cells expressing Sos
4. Wash and elute bound proteins
5. Run eluted proteins in SDS, transfer to membrane and do a western blot with antibodies against SOS
6. See that Sos binds to Drk

Question 17

GST pulldown - Drk and SevB11

Answer 17

1. Make a GST-Drk fusion protein in bacteria
2. Purify the protein by binding to glutathione beads
3. Incubate with lysates from cells expressing SevB11 (constitutively active; i.e. dimerized and phosphorylated)
4. Wash and elute proteins
5. Do a Western Blot with antibodies against Sev

Question 18

Dpp pathway

Answer 18

1. Dpp diffusible ligand binds to and activates TGBF receptors
   a. The receptors are heterodimers and both subunits are Ser/Thr kinases
2. Ligand binding results in association of type 1 and 2 receptors to form a tetramer
3. Type 2 receptor phosphorylates the type 1 receptor, leading to its activation
4. The type 1 receptor phosphorylates smad 2 or 3
   a. Smad 2/3 are in a conformation whereby they are inactive unless phosphorylated
5. Phosphorylated Smad 2 or 3 are now active and associate with Smad 4, making an oligomer
6. The oligomer translocates into the nucleus and associates with co-transcriptional regulators promoting transcription of dpp target genes (ex: omb and spalt)

Question 19

Enhancer trap

Answer 19

• Method utilizes recombinant transposon construct that is randomly inserted in the genome and that, when inserted near the enhancer sequence of a gene, will express the reporter gene, generally LacZ
• One can identify the normal transcription of the gene by the presence of X-gal
• Tells us something about the tissue specificity and temporal pattern of the gene

Question 20

Engrailed –> Hh pathway –> Dpp

Answer 20

1. Engrailed transcription factor defines the posterior compartment of the wing imaginal disc
2. En activates transcription of Hh in posterior cells while also repressing the transcription of Ci (the effector of the Hh pathway) in these cells
3. Hh diffuses to nearby cells, posterior (engrailed expressing cells) do not respond to Hh binding to Ptc receptor but the nearby adjacent stripe does, leading to full length Ci promoting dpp expression
4. Dpp diffuses away to nearby cells and binds to the dpp receptor, promoting the expression of its target genes

Question 21

Hh pathway without Hh

Answer 21

1. In the absence of the Hedgehog signal, Patched inhibits Smoothed by preventing its release from intracellular vesicles
2. Ci transcription factor is sequestered in the cytoplasm by Cos2 which brings it to microtubules to which cos2 binds
3. Ci gets phosphorylated by PKA and other kinases (CK1 and GSK3)
4. Phosphorylated Ci gets recognized by a ubiquitin ligase leading to its ubiquitination
5. Ubiquitinated Ci gets cleaved in the proteasome to make a smaller, cleaved Ci
6. Cleaved Ci translocates into the nucleus and acts as a transcriptional repressor of Hh target genes

Question 22

Hh pathway in presence of Hh

Answer 22

1. Hedgehog binds to its receptors, Ihog and Patched
2. Hedgehog binding results in internalization and downregulation of patched
   a. Patched gets taken in by receptor mediated endocytosis using clathrin coated vesicles leading to its degradation in the lysosome
3. Inhibition of smoothened is relieved and the intracellular vesicle fuses with the membrane, leading to Smoothened becoming embedded in the plasma membrane
4. Smoothened gets phosphorylated by PKA
5. Phosphorylated Smoothened recruits Cos2 to the cell membrane and Cos2 brings Ci to the cell surface
6. Cos2 is unable to recruit PKA and other kinases to phosphorylate Ci
7. Ci doesn’t get phosphorylated, ubiquitinated or cleaved
8. Full length Ci is released from Cos2 and enters the nucleus where it promotes the transcription of Hh target genes

Question 23

Making mutant clones

Answer 23

• Start with individual fruit fly that is heterozygous for the dpp null mutation, one homologue has dpp- and the other has dpp+, dpp is a recessive mutation
• The other homologue, has the GFP transgene and both homologues have FRT sites
• Express FLP recombinase to get recombination between chromosomes
• At mitosis, get segregation leading to getting homozygous mutant dpp with no GFP and homozygous wt with two GFP copies
• These cells divide and produce clone of cells

Question 24

FLPout technique

Answer 24

• Have genetically engineered chromosome that has actin promoter, FRT site, GFP, stop codon, FRT, dpp-
• Under normal conditions, dpp is expressed endogenously in normal area but the mutation is not expressed due to the stop codon, transcript not translated past GFP
• Then add flipase to induce recombination leading to excision of GFP, the stop codon and an FRT site
• Absence of GFP indicates which cells are overexpressing dpp
o Get clone of cells in patches
o Mis/overexpression

Question 25

Wnt pathway in absence of Wnt.

Answer 25

1. In the absence of the Wnt signal, Frizzled and LRP co-receptors are inactive
2. B-catenin gets phosphorylated by the kinases of the destruction complex (kinases = CK1 and GSK3, structural components = Axin, APC)
3. Phosphorylated B-catenin is recognized by ubiquitin ligase which adds a poly-u chain, leading to proteasomal degradation
4. In the absence of B-catenin, TCF represses the transcription of Wnt target genes

Question 26

Wnt pathway in presence of Wnt signal

Answer 26

1. Secreted Wnt binds to Frizzled and LRP co-receptors
2. LRP and Frz are activated leading to Dishevelled activation
3. Activated Dishevelled binds axin brining it to the activated receptor
4. GSK3 and CK1 phosphorylate LRP
5. Axin binds the phosphorylated LRP and is inactivated
6. Axin inactivation results in disassembly of the destruction complex
7. B-catenin is no longer degraded and is free to enter in the nucleus
8. B-catenin binds to TCF to stimulate transcription of Wnt target genes

Question 27

Familial APC

Answer 27

1. Person has inherited a mutant allele from either parent for APC
2. Chance mutation in the wt allele leads to loss of heterozygosity in a single cell in the gut
3. Destruction complex is non-functional
4. B-catenin doesn’t get dedgraded and binds to TCF to promote transcription of Wnt target genes, such as myc
5. Myc promotes cell division
6. Cells continues to divide even after loss of Wnt signal from the crypt
7. Result is multiple polyps and possible cancer

Question 28

Notch pathway

Answer 28

1. Delta transmembrane ligand binds to Notch, triggering cleavage
2. Notch gets cleaved 3 different times and one of these involves Presinilin
3. Cleavage of Notch results in the formation of the NICD that is free from the membrane
4. NICD enters the nucleus dependent on its nls and binds to co-transcription factors
5. Induces transcription of Hairy

Question 29

Lateral inhibition and Notch

Answer 29

1. Mash1 and Ngn2 transcription factors promote transcription of Delta (Dll1)
2. Delta binds Notch leading to its cleavage at three sites, one of which involves Presinilin resulting in NICD
3. NICD translocates into the nucleus dependent on its nls and binds to co-transcription factors to promote transcription of Hairy
4. Hairy represses the transcription of Ngn2 and genes required for neural fate
5. Low mash1 and ngn2 expression levels results in lower Delta expression, typical of cell adopting epidermal fate

Question 30

Wnt and Notch signaling in the intestinal crypt

Answer 30

1. Wnt is secreted from Paneth cells in the intestinal crypt (non-dividing differentiated cells) and promotes myc expression in stem cells which divide to form transit-amplifying cells
2. Transit amplifying cells divide rapidly and move away from the crypt
3. These cells stop dividing as they are pushed up the crypt away from the Wnt signal
4. Wnt pathway activation promotes Delta expression and thereby the Notch pathway in transit amplifying cells
5. Activation of Notch leads to absorptive fate
6. Secretory fate is the default fate, the cell with notch active is the loser and adopts the absorptive fate