||<p>Inositol phosphates (IPs) are versatile metabolites that play important roles in
multiple cellular processes. They have been considered signaling messengers that relay
extracellular signals via a wave of their production and allosteric regulation of
downstream targets. In addition to this classical role, recent studies have revealed
that certain IPs can also function as protein structural cofactors. However, except
for the two plant hormone receptors TIR1 and COI1, these IP binding proteins have
neither sequences nor functions in common. Therefore, to test whether other cellular
proteins are also subjected to this type of regulation and whether an IP binding motif
exists, more proteins that bind IPs in a similar manner need to be identified. Via
a proteome-wide biochemical screen, two yeast proteins were found to contain IP<sub>6</sub>
as an integral component. One is the N-terminal acetyltransferase A complex (NatA),
and the other one is Tif31 (or Clu1). IP<sub>6</sub> binding was also observed in
NatC, another N-terminal acetyltransferase. The bioinformatics analysis and mutagenesis
study showed that tandem tetratricopeptide repeats (TPRs), the only common structural
element of NatA and Tif31, were responsible for coordinating IP<sub>6</sub>. This
mechanism of IP<sub>6<sub> binding is conserved in the fly homologs of these proteins.
</p><p>NatA is one of the enzymes that acetylate the α-amino groups at protein
N-termini. This widespread protein modification affects a wide range of cellular processes.
IP<sub>6</sub> was shown to be essential for yeast NatA <italic>in vitro</italic>
thermostability and for some but not all functions of the protein in cells grown under
temperature stress. Other multiple phosphate-containing molecules including IP<sub>5</sub>
species and the bacterial alarmone ppGpp were found to bind NatA and partially compensate
for the lack of IP<sub>6</sub>. IP<sub>6</sub> also binds the human NatA homolog.
This binding is crucial for hNatA complex formation, <italic>in vitro</italic> and
<italic>in vivo</italic> activities, and ability to rescue NatA-deficient phenotypes
when it is expressed in yeast. Therefore, IP<sub>6</sub> acts as a molecular glue
that brings hNatA (and hNatE) subunits together. The other protein found in our screen,
Tif31, is important for normal mitochondrial morphology and distribution. In cells
that cannot produce IP<sub>4</sub>, IP<sub>5</sub> and IP<sub>6</sub>, Tif31 levels
were significantly decreased and these cells exhibited severe mitochondrial aggregation.
Tif31 mutants that cannot bind IP<sub>6</sub> showed a reduction in cellular levels,
a shift to high molecular weight complexes or aggregates, and inability to rescue
<italic>tif31</italic>δ mitochondrial phenotype. This study established the
vital role of IP<sub>6</sub> and IP<sub>5</sub> in maintaining Tif31 stability and
Tif31-mediated regulation of mitochondrial distribution.</p><p>Collectively, this
dissertation discovered two proteins that use IP<sub>6</sub> as a structural cofactor.
For the first time, a conserved IP<sub>6</sub> binding motif has been shown to be
present in certain TPR-containing proteins. Via tight binding to these proteins, IP<sub>6</sub>
stabilizes their structures or subunit interaction. This research provides mechanistic
evidence for the interplay between IP biology and N-terminal acetylation as well as
between IP biology and mitochondrial morphology.</p>