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The endosomal sorting complex required for transport (ESCRT) plays a crucial role in the transportation and degradation of proteins. We determined that Vps27, a key protein of the ESCRT-0 complex, is required for the transport of the virulence factor laccase to the cell wall in Cryptococcus neoformans. Laccase activity was perturbed, as was melanin production, in vps27Δ strains. In the absence of VPS27, there was an accumulation of multivesicular bodies with vacuolar fragmentation and mistargeting of the vacuolar carboxypeptidase CPY/Prc1, resulting in an extracellular localization. In addition, deletion of VPS27 resulted in a defect in laccase targeting of a Lac1-green fluorescent protein (GFP) fusion to the cell wall with trapping within intracellular puncta; this deletion was accompanied by reduced virulence in a mouse model. However, the actin cytoskeleton remained intact, suggesting that the trafficking defect …