Identification of Novel Factors Involved in Vesicle Formation at the Multivesicular Body in Yeast...
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Transcript of Identification of Novel Factors Involved in Vesicle Formation at the Multivesicular Body in Yeast...
Identification of Novel Factors Involved in Vesicle Formation at the
Multivesicular Body in Yeast
Erika Hanson
Stevens Lab
Protein Pathways in Cells
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.
Raiborg and Stenmark Nature, 2009
MVB sorting machinery is involved in MVB biogenesis, cytokinesis, and viral budding
ESCRT( Endosomal Sorting Complex required for transport) complexes concentrate cargo at MVB
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.
Q:How do vesicles on the MVB form?Are there other proteins
besides the ESCRT complexes involved?
Genetic screen for yeast MVB components
Normal MVB
MVB defect
No GrowthOn - His media
Growth on-His media
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
?
How Does Ubx2p Affect Sorting at the MVB?
Plasma Membrane
Lysosome(Vacuole)MVB
Nucleus
ER
Golgi
??
Ubx2p
Is Ubx2 required for ESCRT association with MVBs?
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
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
ESCRT assembly localization
Curtiss and Jones 2006
A B C
A. Normal ESCRT assembly
B. ESCRT can’t disassemble
C. ESCRT can’t assemble
Wild type
ESCRT mutant
Loss of Ubx2p
Endosome localization
AberrantMVB
ESCRT 0 assembly occurs in absence of Ubx2p
DIC GFP Merge
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
Acknowledgements
•My mentor, Emily Coonrod•Laurie Graham•Tom Stevens•Amy Stein•Stevens Lab•Peter O’Day•SPUR program