Browsing by Author "Kozubowski, Lukasz"

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Open Access
Changes in Bni4 localization induced by cell stress in Saccharomyces cerevisiae
(2010) Larson, Jennifer R; Kozubowski, Lukasz; Tatchell, Kelly
Septin complexes at the bud neck in Saccharomyces cerevisiae serve as a scaffold for proteins involved in signaling, cell cycle control, and cell wall synthesis. Many of these bind asymmetrically, associating with either the mother-or daughter-side of the neck. Septin structures are inherently apolar so the basis for the asymmetric binding remains unknown. Bni4, a regulatory subunit of yeast protein phosphatase type 1, Glc7, binds to the outside of the septin ring prior to bud formation and remains restricted to the mother-side of the bud neck after bud emergence. Bni4 is responsible for targeting Glc7 to the mother-side of the bud neck for proper deposition of the chitin ring. We show here that Bni4 localizes symmetrically, as two distinct rings on both sides of the bud neck following energy depletion or activation of cell cycle checkpoints. Our data indicate that loss of Bni4 asymmetry can occur via at least two different mechanisms. Furthermore, we show that Bni4 has a Swe1-dependent role in regulating the cell morphogenesis checkpoint in response to hydroxyurea, which suggests that the change in localization of Bni4 following checkpoint activation may help stabilize the cell cycle regulator Swe1 during cell cycle arrest.
Open Access
Relative Contributions of Prenylation and Postprenylation Processing in Cryptococcus neoformans Pathogenesis.
(mSphere, 2016-03) Esher, Shannon K; Ost, Kyla S; Kozubowski, Lukasz; Yang, Dong-Hoon; Kim, Min Su; Bahn, Yong-Sun; Alspaugh, J Andrew; Nichols, Connie B
Prenyltransferase enzymes promote the membrane localization of their target proteins by directing the attachment of a hydrophobic lipid group at a conserved C-terminal CAAX motif. Subsequently, the prenylated protein is further modified by postprenylation processing enzymes that cleave the terminal 3 amino acids and carboxymethylate the prenylated cysteine residue. Many prenylated proteins, including Ras1 and Ras-like proteins, require this multistep membrane localization process in order to function properly. In the human fungal pathogen Cryptococcus neoformans, previous studies have demonstrated that two distinct forms of protein prenylation, farnesylation and geranylgeranylation, are both required for cellular adaptation to stress, as well as full virulence in animal infection models. Here, we establish that the C. neoformans RAM1 gene encoding the farnesyltransferase β-subunit, though not strictly essential for growth under permissive in vitro conditions, is absolutely required for cryptococcal pathogenesis. We also identify and characterize postprenylation protease and carboxyl methyltransferase enzymes in C. neoformans. In contrast to the prenyltransferases, deletion of the genes encoding the Rce1 protease and Ste14 carboxyl methyltransferase results in subtle defects in stress response and only partial reductions in virulence. These postprenylation modifications, as well as the prenylation events themselves, do play important roles in mating and hyphal transitions, likely due to their regulation of peptide pheromones and other proteins involved in development. IMPORTANCE Cryptococcus neoformans is an important human fungal pathogen that causes disease and death in immunocompromised individuals. The growth and morphogenesis of this fungus are controlled by conserved Ras-like GTPases, which are also important for its pathogenicity. Many of these proteins require proper subcellular localization for full function, and they are directed to cellular membranes through a posttranslational modification process known as prenylation. These studies investigate the roles of one of the prenylation enzymes, farnesyltransferase, as well as the postprenylation processing enzymes in C. neoformans. We demonstrate that the postprenylation processing steps are dispensable for the localization of certain substrate proteins. However, both protein farnesylation and the subsequent postprenylation processing steps are required for full pathogenesis of this fungus.