Identification of Novel Factors Involved in Vesicle Formation at the Multivesicular Body in Yeast Erika Hanson Stevens Lab.

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