BINDING INTERACTIONS BETWEEN MIT DOMAINS AND ESCRT-III PROTEINS

Matthew Miller

Abstract


The ESCRT-III (Endosomal Sorting Complexes Required for Transport) proteins play crucial roles in controlling abscission (the process of separating daughter cells in cytokinesis) (Figure 1) and other cellular processes such as HIV budding that involve membrane fission. The ESCRT pathway is a crucial part of cellular function in all eukaryotic organisms(1). ESCRT-III proteins provide the constricting force required for membrane fission, and they recruit essential cofactors that contain Microtubule Interacting and Trafficking (MIT) domains. An example of a crucial role that the ESCRT machinery plays is that the machinery reseals the nuclear envelope during anaphase(1).

We have studied how the ESCRT pathway functions by performing binding experiments with the greater than 20 known MIT domains that interact with the ESCRT pathway, which will provide a knowledge of the processes in which these proteins function. This will help us understand the process by which HIV takes advantage of the body’s ESCRT machinery that facilitates cell division, as well as how cell division occurs.

We used biochemical techniques such as protein expression, protein purification, size exclusion chromatography, ion exchange chromatography and fluorescence polarization to identify how the MIT domains bind with the various Microtubule Interacting Motifs (MIMs) of the ESCRT-III pathway. MIMs are the C-terminal tails of ESCRT-III proteins that facilitate interactions between the ESCRT machinery and MIT domain-containing proteins.

We discovered that MIT domain-containing proteins have very different binding specifities. An example of this would be Katanin, which only interacts with CHMP3 in comparsion with MITD1 that has a much broader binding specificity and interacts with CHMP1A, CHMP1B, CHMP2A, CHMP2B, CHMP3, CHMP6, and IST1. The knowledge that Katanin only interacts with CHMP3 raises a lot of new questions such as how these proteins interact, and why does it only interact with CHMP3.


Keywords


ESCRT pathway, MIT domains, fluorescence anisotrophy

Full Text: PDF

Refbacks

  • There are currently no refbacks.