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 Growth On - 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 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 Ubx2p Method: 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 AB C A.Normal ESCRT assembly B.ESCRT can’t disassemble C.ESCRT can’t assemble

Wild type ESCRT mutant Loss of Ubx2p Endosome localization Aberrant MVB ESCRT 0 assembly occurs in absence of Ubx2p DICGFPMerge

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