Polarized hyphal growth dominates the life cycle of filamentous fungi and is essential to disease progression for many fungal pathogens. Despite its importance, much of the basic biology controlling the process remains to be elucidated. Protein Nmyristoylation is one process important to hyphal growth for which the direct mechanism for this connection is not understood. N-myristoylation is mediated by Nmyristoyltransferase (NMT), which links 14-carbon myristate to target proteins. In Aspergillus nidulans, a mutation in the NMT gene (swoF1) results in abnormal morphogenesis during spore germination and the establishment of hyphal polarity. I hypothesize that a protein or proteins downstream of NMT are important for polarized hyphal growth. Using a forward genetic approach, I obtained six suppressors of swoF1. I found that three were proteasome-related and a mutation in genes encoding 26S proteasome subunits by-passed the polarity defects of swoF1. Interestingly, N-myristoylation negatively regulated the activity of the 26S proteasome. This result was confirmed by treating with the proteasome inhibitor MG132. This is the first finding of a connection between N-myristoylation and proteasome function during polarized growth. To identify targets by reverse genetic analysis, I found that 41 proteins (of more that 10,000 encoded by the organism) were predicted to be myristoylated in silico. Three were ADP ribosylation factors (ARF), proteins known to be involved in vesicle formation and trafficking in other systems. I chose ArfA (AN1126.3), ArfB (AN5020.3), and ArlA (AN5912.3) for further characterization of polarization in this study. ArfA::GFP discretely localized to endomembrane likely to be Golgi bodies. ArfB::GFP localized to septa and plasma membrane. N-myristoylation determined the localization of both ArfA and ArfB. Disruption of the arfB gene resulted in loss of polarity establishment and endocytosis. Together these results suggest that endocytosis plays an important role in maintaining hyphal polarized growth and in shaping the cell apex.

As there is a need for fully validated drug targets in <i>Trypanosoma cruzi</i>, the genetic andbiochemical essentiality of <i>N</i>-myristoyltransferase (NMT) was assessed. The geneticrequirement was assessed using a classical gene replacement strategy, attempting tosequentially replace the endogenous alleles with drug resistance genes to generate an<i>NMT</i> null parasite. It was only possible to achieve this in the presence of an ectopiccopy of <i>NMT</i> under constitutive expression, providing the strongest evidence that thisgene is essential for the proliferation of the epimastigote. While both NMT and <i>N</i>-myristoylationwere detected in all lifecycle stages, there were subtle differences in theexpression of several myristoylated proteins. However, at least ~10 myristoylatedproteins were common throughout the lifecycle. In addition, <i>N</i>-myristoylation in thisparasite was found to be primarily associated with nascent protein synthesis, astreatment with cycloheximide reduced the number of <i>N</i>-myristoylated proteins detected. The sensitivity of epimastigotes to the inhibitor DDD85646 correlated with theexpression of NMT, suggesting it to be the target in the parasite. This was confirmedby the dose-dependent depletion of <i>N</i>-myristoylated proteins detected in parasitestreated with this compound. Mechanism of action studies revealed a cytokinesis defectcaused by the inhibition of <i>N</i>-myristoylation and NMT. Overexpression of NMT wasable to rescue these cells from this phenotype confirming that it is NMT mediated. The<i>N</i>-myristoylated proteins comprising the <i>N</i>-myristoylome of the epimastigote wereidentified using the myristic acid analog, azidomyristate and a chemical proteomicsapproach. Combining label-free and SILAC methodologies, 38 proteins were enrichedfrom azidomyristate labelled cells, 35 of which were predicted to have a glycine afterthe initial methionine. The findings from these experiments have led to the mostcomprehensive <i>N</i>-myristoylome of <i>T. cruzi</i> studied to date and provide severalhypotheses, by which the inhibition of NMT leads to the observed cytokinesis defect.

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