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![Page 1: Identification of Novel Factors Involved in Vesicle Formation at the Multivesicular Body in Yeast Erika Hanson Stevens Lab.](https://reader036.fdocuments.net/reader036/viewer/2022062515/56649f445503460f94c65561/html5/thumbnails/1.jpg)
Identification of Novel Factors Involved in Vesicle Formation at the
Multivesicular Body in Yeast
Erika Hanson
Stevens Lab
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Protein Pathways in Cells
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Multivesicular Body(MVB)
• Formed by the budding of the limiting endosomal membrane and the formation of lumenal vesicles
• Vesicles are formed by the inward budding of the early endosome membrane
• Proteins are sorted at these vesicles.
• The MVB eventually fuses with the vacuole, and endocytosed proteins are degraded.
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Raiborg and Stenmark Nature, 2009
MVB sorting machinery is involved in MVB biogenesis, cytokinesis, and viral budding
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ESCRT( Endosomal Sorting Complex required for transport) complexes concentrate cargo at MVB
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ESCRT machinery
Rieder and Banta 1996
• The ESCRT pathway is highly conserved in eukaryotes
• Necessary for the formation of the vesicles on the MVB
• Vesicle formation is essential to protein trafficking in cells through the MVB
• Defects in ESCRT complexes produce an aberrant MVB.
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Q:How do vesicles on the MVB form?Are there other proteins
besides the ESCRT complexes involved?
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Genetic screen for yeast MVB components
Normal MVB
MVB defect
No GrowthOn - His media
Growth on-His media
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Ubx2• Integral
membrane protein in the Endoplasmic reticulum(ER)
• Required for the ER associated degradative process.
• Cells lacking Ubx2p form aberrant MVBs
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Neuber and Jarosch 2005
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?
How Does Ubx2p Affect Sorting at the MVB?
Plasma Membrane
Lysosome(Vacuole)MVB
Nucleus
ER
Golgi
??
Ubx2p
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Is Ubx2 required for ESCRT association with MVBs?
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Goal:Determine if ESCRT complexes assemble in the absence
of Ubx2pMethod:
• Use fluorescence microscopy to analyze ESCRT assembly in absence of Ubx2p
• GFP tag components of ESCRT complexes and see where localize in cell when Ubx2p is absent
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Approach
• Vps24-GFP, ESCRT-III• vps46-GFP, ESCRT-III
and vps4 • vps27-GFP, ESCRT 0
• Examine localization in wild type cells and cells with out Ubx2p
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ESCRT assembly localization
Curtiss and Jones 2006
A B C
A. Normal ESCRT assembly
B. ESCRT can’t disassemble
C. ESCRT can’t assemble
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Wild type
ESCRT mutant
Loss of Ubx2p
Endosome localization
AberrantMVB
ESCRT 0 assembly occurs in absence of Ubx2p
DIC GFP Merge
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Summary
•Ubx2p doesn’t affect ESCRT 0 assembly
Future Directions:
•Look at assembly of all ESCRT components in the absence of Ubx2p•Finish making reagents to visualize vps24-GFP and vps46-GFP localization in absence of Ubx2p
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Acknowledgements
•My mentor, Emily Coonrod•Laurie Graham•Tom Stevens•Amy Stein•Stevens Lab•Peter O’Day•SPUR program