Novel Ist1-Did2 Complex Functions at a Late Step in MVB Sorting

Sarah M. Rue,* ${dagger}$ ${ddagger}$ Sara Mattei, ${dagger}$ ${sect}$ Suraj Saksena,||¶ and Scott D. Emr* ${sect}$ ||¶

^*Department of Cellular and Molecular Medicine, Division of Biological Sciences, and ^||Howard Hughes Medical Institute, University of California San Diego, La Jolla, CA 92093

Monitoring Editor: Sean Munro

In S. cerevisiae, integral plasmamembrane proteins destined for degradation and certain vacuolarmembrane proteins are sorted into the lumen of the vacuole viathe multivesicular body (MVB) sorting pathway, which dependson the sequential action of three endosomal sorting complexesrequired for transport (ESCRTs). Here, we report the characterizationof a new positive modulator of MVB sorting, Ist1. We show thatendosomal recruitment of Ist1 depends on ESCRT-III. Deletionof IST1 alone does not cause cargo sorting defects. However,synthetic genetic analysis of double mutants of IST1 and positivemodulators of MVB sorting showed that ist1 ${Delta}$ is synthetic withvta1 ${Delta}$ and vps60 ${Delta}$ , indicating that Ist1 is also a positive componentof the MVB sorting pathway. Moreover, this approach revealedthat Ist1-Did2 and Vta1-Vps60 compose two functional units.Ist1-Did2 and Vta1-Vps60 form specific physical complexes, and,like Did2 and Vta1, Ist1 binds to the AAA-ATPase Vps4. We provideevidence that the ist1 ${Delta}$ mutation exhibits a synthetic interactionwith mutations in VPS2 (DID4) that compromise the Vps2-Vps4interaction. We propose a model in which the Ist1-Did2 and Vta1-Vps60complexes independently modulate late steps in the MVB sortingpathway.

These authors contributed equally to this work.

Present addresses: Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121; ^¶Cornell Institute for Cell and Molecular Biology, Cornell University, 307 Biotechnology Building, Ithaca, NY 14853.

Address correspondence to: Scott D. Emr (sde26{at}cornell.edu)