Regulated proteolysis of green fluorescent protein fusion proteins in Aspergillus fumigatus

Abstract

Aspergillosis caused by Aspergillus fumigatus is a clinical issue of such severity that the World Health Organization has designated this organism as 1 of the 4 most critical fungi to study. Progress in A. fumigatus has been limited by the availability of genetic tools with which to study this filamentous fungus. Currently available means of altering the dosage of genes and gene products include construction of disruption mutants as well as regulated promoters. These are powerful techniques but somewhat limited for the analysis of essential genes. Here we describe a new method that permits regulated proteolysis of any A. fumigatus protein that can be made as a fusion protein to the well-described Green Fluorescent Protein (GFP) of Aequorea victoria. A GFP fusion protein of interest can be targeted for degradation using a single-chain antibody called a nanobody that recognizes GFP (GFPNb). This GFPNb is in turn fused to an E3 ligase protein called Rnf4 from rat that efficiently ubiquitinates target proteins. A fusion gene was constructed under control of a doxycycline-inducible promoter that produced a GFPNb-Rnf4 fusion protein in A. fumigatus. Here we show that production of this GFPNb-Rnf4 protein led to the rapid proteolysis of a variety of GFP fusion proteins. Additionally, we found that some GFP fusion proteins triggered a corresponding genomic response when their degradation was induced while others were simply degraded. These studies provide a new means to directly regulate protein levels in A. fumigatus and generate new alleles of genes exposing the underlying regulatory circuitry. ImportanceAspergillus fumigatus is the major filamentous fungal pathogen of such importance in infectious disease that it has been designated as one of the 4 most critical fungal organisms to study. One of the limiting capabilities in the analysis of A. fumigatus is the limiting genetic toolbox that can be employed in this fungus. Here we describe the first system that allows the regulated degradation of a protein of interest using a recombinant single protein chain antibody (nanobody) directed against Green Fluorescent Protein (GFP). GFP is routinely used to construct fusion proteins that in turn allow localization of the fluorescent protein and immunological detection. Here we use this nanobody to deliver a ubiquitin ligase that in turn causes rapid depletion of GFP fusion proteins. This allows direct perturbation of protein levels in A. fumigatus, a feature not previously accessible to experimentation.