Novel Ist1-Did2 complex functions at a late step in multivesicular body sorting

Area of Science:

• Cell Biology
• Molecular Biology
• Protein Trafficking

Background:

• The multivesicular body (MVB) pathway is essential for sorting specific proteins to the vacuole in Saccharomyces cerevisiae.
• This pathway relies on endosomal sorting complexes required for transport (ESCRT) machinery for protein sorting.
• Integral plasma membrane proteins and vacuolar membrane proteins are key cargo sorted via the MVB pathway.

Purpose of the Study:

• To characterize a novel positive modulator of the MVB sorting pathway, named Ist1.
• To elucidate the role of Ist1 in protein sorting and its dependence on other MVB pathway components.
• To investigate the functional and physical interactions of Ist1 with other MVB sorting factors.

Main Methods:

• Characterization of Ist1's endosomal recruitment, dependent on ESCRT-III.
• Synthetic genetic analysis of double mutants involving IST1 and other MVB pathway modulators (Vta1, Vps60).
• Co-immunoprecipitation assays to identify physical complexes (Ist1-Did2, Vta1-Vps60) and interaction with Vps4.
• Analysis of synthetic interactions between ist1Δ and vps2 (did4) mutations affecting Vps2-Vps4 interaction.

Main Results:

• Endosomal recruitment of Ist1 is dependent on ESCRT-III.
• Deletion of IST1 alone does not impair cargo sorting, but synthetic interactions with vta1Δ and vps60Δ reveal Ist1's positive role.
• Ist1-Did2 and Vta1-Vps60 form distinct functional and physical complexes that interact with the AAA-ATPase Vps4.
• The ist1Δ mutation shows synthetic interaction with vps2 mutations that disrupt Vps2-Vps4 interaction.

Conclusions:

• Ist1 acts as a positive component in the MVB sorting pathway, modulating late steps.
• The MVB sorting pathway involves at least two functional units: Ist1-Did2 and Vta1-Vps60.
• These complexes likely function independently to regulate the final stages of MVB formation and cargo sorting.