committee meeting 29oct2012-3starklab.slu.edu/jce/shancmteoct2012.pdfsummary* * •...
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Mechanism of protein sor0ng in Drosophila
Shan Luan 29 Oct 2012
Receptor-‐dependent protein sor0ng
Maria Francisca Cou0nho, Maria João Prata, Sandra Alves, (2012) Mannose-‐6-‐phosphate pathway: A review on its role in lysosomal func0on and dysfunc0on. Molecular Gene1cs and Metabolism, Volume 105, Issue 4, Pages 542-‐550
From: Boman, A.L. (2001) J. Cell Sci. 114: 3413-‐3418
Model for cargo retrival • A unique adaptor protein is -‐ iden0fied in yeast two hybrid protein interac0on with ADP ribosyla0on factor-‐binding 3 (ARF3) -‐ iden0fied by expressed sequence tag (EST) database for genes encoding proteins with VHS (Vps27p, Hrs and STAM) and ear domain. • Monomeric clathrin adaptors
• GGA: Golgi-‐localized, gamma-‐ear-‐containing, ARF-‐binding
GGAs
• 2 GGA family proteins in yeast (not essen0al) • 3 GGA family proteins in mammals (RNAi in cultured cells-‐not essen0al) • 1 GGA family protein in nematodes (not essen0al) • 1 GGA family protein in flies (no muta6ons were established, RNAi knockdown showed lethality)
Research Ques0on: Is GGA essen0al in Drosophila? What is GGA essen0al for?
P-‐element excision-‐mediated muta0on
GGA mutant allele
GGA
[ ]
Lethal maps between 8C8 and 8C12, 500 kb to the lee of GGA.
Dp(1;3)DC203 [8C4-‐8C13]
Mapping lethality in GGA muta0on
Remove lethal muta0on from GGAp1
y sn v g f
sn+ y+ L GGAP1 v+ g+ f+
sn y L+ GGA+ v g f
GGAp1 allele
Mapping allele
Screen for sn and v+ males by PCR
GGA fwd GGA rev
9 10 11 12 15 1 2 3 4 5 6 7 8 GGAp1 wt Wt 4470bp GGAp1 3270bp
GGAP1 does not cause age-‐dependent re0nal degenera0on.
Wild type " ey/GMR-Gal4; dGGA RNAi2, "
GGAP1;Dp(1;3)DC203" w1118;Dp(1;3)DC203"
Adult flies were aged 14 days at 25C.
Is GGAP1 a null muta0on?
Genera0on of a Targeted Knockout Allele of dGGA
Screen for GGA null allele
PXH up 85 135
PXH down 85 135
GGA 2200 Ac0n primers wt 29 70 72 85 112 135 wt 29 70 72 85 112 135
wt GGA
GGA 2200 826bp
Hsp>mini-‐white EYFP
PXH up PXH up PXH down PXH down
1kb
3824bp 3458bp
GGA KO // // // //
GGAΔ are viable and fer0le
Test semi-‐lethality of the GGAΔ and GGAP1 alleles
Crosses F1 ♀ heterozygous
F1 ♂ P-‐Value
☿GGAΔ X ♂GGA+ 229 183 0.5310
☿GGA+ X ♂GGAΔ 223 195
☿GGAP1 X ♂GGA+ 192 174 0.7815
☿GGA+ X ♂GGAP1 259 225
Mini-‐GGA transgene complements GGAΔ flies.
P{GGA+}
dGGA Knockout Muta0on Fails to Rescue RNAi Knockdown of dGGA
☿GGAΔ; Act5C-‐Gal4/CyO X ♂ yw; GGA-‐RNAi2.
Only GGAΔ; +/CyO; +/GGA-‐RNAi2
Suggest previous observed lethality in RNAi Knockdown is off target.
Receptor-‐dependent protein sor0ng
Maria Francisca Cou0nho, Maria João Prata, Sandra Alves, (2012) Mannose-‐6-‐phosphate pathway: A review on its role in lysosomal func0on and dysfunc0on. Molecular Gene1cs and Metabolism, Volume 105, Issue 4, Pages 542-‐550
Cathepsin L and cathepsin D processing are not impaired in GGA null flies.
GGA in lysosomal func0on
1) Compromise lysosome, decrease enzyme efficiency
2) Further reduce GGA 3) Chloroquine in cell leads to lysosomal pH
increase. 4) 10mM Chloroquine is toxic in flies. We fed chloroquine to GGA mutant flies.
GGA mutant flies are hypersensi0ve to dietary chloroquine (20mM).
A: GGA+(5.25 days n=97) B: GGAΔ ;p{GGA+}A2 (4.98 days n=161) C: GGAΔ;p{GGA+}E2 (4.45 days n=151) GGAΔ (2.9 days n=181) GGAΔ (2.9 days n=181) GGAΔ (2.9 days)
P-‐value<0.0001 P-‐value<0.0001 P-‐value<0.0001
Eissenberg JC, Ilvarsonn AM, Sly WS, Waheed A, Krzyzanek V, Pohlmann R, Waschkau D, Kretzchmar D, Dennes AC. Drosophila GGA-‐model: An ul0mate gateway to GGA analysis. Traffic. (2011) 12:1821-‐1838.
Bchs Overexpression
GGA knockout enhances the eye phenotype caused by Bchs overexpression.
P-‐value <0.001
P-‐value <0.05
10 heads per sample and triplicate
GGA mutant flies are hypersensi0ve to amino acid starva0on.
A:GGA+ (13.65 days n=202) B: GGAΔ ;p{GGA+}A2 (14.92 days n=131) C: GGAΔ;p{GGA+}E2 (16.4 days n=144) GGAΔ (11.29 days n=212) GGAΔ (11.29 days n=212) GGAΔ (11.29 days n=212)
P-‐value<0.0001 P-‐value<0.0001 P-‐value<0.0001
Summary • GGA is not essen0al in survial. • First report of a targeted knockout of an X-‐linked gene using Chen (2009)’s
method. • In GGAΔ flies, there is no detectable defect in cathepsinL or cathepsinD
processing. • in GGAP1 or GGAΔ flies, there is no evidence for age-‐dependent re0nal
degenera0on. • GGAΔ flies is hypersensi0e to dietary chloroquine, suppor0ng a model in which
GGA family proteins contribute to lysosomal homeostasis under stress condi0ons.
• GGAΔ with Bchs overexpression flies enhanced the eye phenotype, sugges0ng that dGGA could directly or indirectly be involved in the process of autophagy.
• GGAΔ flies are hypersensi0ve to amino acid starva0on, consistent with impaired autophagy. It is the first hint GGA is required in evolu0on.
Ongoing projects: Proteins poten0ally involved in sor0ng lysosomal hydrolases.
From: Boman, A.L. (2001) J. Cell Sci. 114: 3413-‐3418
Model for cargo retrival
Lysosomal enzyme receptor protein (LERP)
• Homolog to mannose 6-‐phosphate receptors (MPR) in mammals
• Rescue missor0ng of soluable lysosomal enzymes in MPR-‐deficient cells
• However, ligand recogni0on does not depend on mannose 6-‐phosphate.
Research ques0on: Is Lerp essen0al in survival? Does Lerp func0on in sor0ng lysosomal hydrolases or sor0ng others e.g. IGF2? How does Lerp func0on in sor0ng lysosomal hydrolases?
Genera0on of a P-‐element Excision-‐mediated Allele of Lerp
Genera0on of a P-‐element Excision-‐mediated Allele of Lerp
w1118; Mi{ET1}LerpMB05321 x yw; CyO, HoP2/Bc1
w1118; CyO, HoP2; Mi{ET1}LerpMB05321 ♂
♂
yw; Bl1/TM3Ser x
☿
☿
Collect male or female: Lerp?/TM3Ser for PCR screening
Genera0on of a Targeted Knockout Allele of LERP
AP-‐1 • Adaptor protein complex 1 (AP-‐1) is a heterotetramer composed of
AP1ϒ, AP1β, AP1μ1, AP1δ1. • the ‘ear’ domain of the γ-‐subunit of AP-‐1 shares homology to the C-‐
terminal GAE (γ-‐adap0n ear) domain of GGA and interacts with clathrin.
• Muta0ons in GGA are not lethal. • Transposable element inser0on at AP-‐1 gamma is lethal. Previous literatures suggested two hypothesis: 1. GGA and AP-‐1 func0on redundantly in sor0ng lysosomal hydrolases. 2. GGA binds receptors on Golgi and hands off the proteins to AP-‐1.
Ap-‐1γ
w1118;Dp(1;3)DC203 [8C4-‐8C13]
Is Ap-‐1γ lethality due to Ap-‐1γ muta0on?
PBac {w+=RB} Ap-‐1γ Y
; Dp(1:3)DC(203) + are viable.
Genera0on of a P-‐element Excision-‐mediated Allele of AP-‐1 gamma
Genera0on of a P-‐element Excision-‐mediated Allele of AP-‐1 gamma
w[1118] PBac{w[+mC]=RB}AP-‐1gamma[e04546]/FM7ywaB
w[1118] PBac{w[+mC]=RB}AP-‐1gamma[e04546]/FM7c x FM7ywaB; CyO, HoP2/+
FM7ywaB ♂
♂
x
☿
☿
Collect male or female: AP-‐1gamma?/FM7ywaB for PCR screening
Thank you! Biochemistry & Molecular Biology Saint Louis University School of Medicine Joel C. Eissenberg Anne M. Ilvarsonn Abdul Waheed Medina Kran0c
Biology Department Saint Louis University William S. Stark
Remove lethal muta0on from GGAp1 y sn v g f
♂ y1sn3 v1 g2 f1 ☿ y, l, GGAp1; Dp(1:3)DC(203) X
y1 sn3 v1 g2 f1 y, l, GGAp1
☿ X ♂ yw ;Dp(1:3)DC(203)
♂ y1sn3 v+ g+ f+; Dp(1:3)DC(203) or + +
X y,l,GGAp1 FM7�GFP ☿
y,l,GGAp1 y1sn3 GGAp1? v+ g+ f+ ☿ ; +
Dietary chloroquine impairs lysosome-‐mediated proteolysis.
LAMP-GFP on normal foodLAMP-GFP on food + 10 mM chloroquine (36 h)