The roles of N-myristoylation in cell morphogenesis in Aspergillus nidulans

Abstract

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.