Project Summary/Abstract
Ubiquitination is an important mechanism that regulates nearly every biological process, and
defects in the ubiquitin system can lead to diseases such as cancer and neurodegeneration. Due to
their role in reversing and regulating ubiquitination, deubiquitinating enzymes have emerged as critical
cell regulators and promising drug targets. Thus, it is imperative to gain a thorough understanding of
the function and regulation of deubiquitinating enzymes.
The goal of this study is to elucidate the function and regulation of MINDY, a newly discovered
family of deubiquitinating enzymes that are K48-chain specific with unclear biological functions. In our
preliminary studies, we find that a pool of MINDY in yeast (dubbed Miy1) is associated with plasma
membrane. Interestingly, disrupting the MIY1 gene leads to a clear increase in plasma membrane
protein ubiquitination and a marked destabilization of a transmembrane protein. We hypothesize that
MINDY family of enzymes are key regulators for the ubiquitination of membrane proteins. To test our
hypothesis and to achieve our objectives, three specific aims are proposed.
Aim 1: What is the biological function of Miy1? We hypothesize that Miy1 maintains proper
mono-ubiquitination of plasma membrane proteins by reversing the erroneously formed K48-linked
ubiquitin chain on these proteins. To test this, we will examine how Miy1 affects ubiquitination of well-
established plasma membrane protein substrates. We will also identify proteins whose ubiquitination
pattern is altered in the MIY1-disruptive mutants. For any identified candidate, we will use in vitro and
in vivo methods to determine how Miy1 affects their level, stability, and subcellular localization.
Aim 2: How is Miy1 regulated? In our preliminary studies, we find that Miy1 undergoes both
phosphorylation and sumoylation. Our hypothesis is that Miy1 is regulated by these post-translational
modifications. To test this, we will identify the modification sites and responsible enzymes, generate
mutants that block the modifications, and examine the effects of blocking these modifications on the
activity and function of Miy1. Alternatively, Miy1 may be regulated via its binding partners. To test this,
we will identify Miy1-interacting proteins and examine their roles in regulating the function of Miy1.
Aim 3: Are the function and regulation of Miy1 conserved? The human homolog of Miy1 is
MINDY-1. Given the highly conserved nature of cell regulation across evolution, it is highly likely that
what we have learned from Miy1 in yeast can be extended to MINDY-1 in humans. To test this, we will
determine if MINDY-1 similarly modulates ubiquitination of plasma membrane proteins; we will also
investigate if the activity and function of MINDY-1 is regulated by phosphorylation and sumoylation as
well as its interacting proteins, in a manner similar to Miy1.