(F) Western blots of peroxisomal fraction (lane 1) and mitochondrial fraction (lane 2) proteins from P. brasiliensis yeast cells were probed with anti-PbMLSr click here antibody. Molecular mass markers are indicated at the side. Detection of PbMLS on cell wall extracts, culture filtrate, crude extract and peroxisomal fraction To determine the subcellular distribution of PbMLS, cell wall-enriched, secreted, PARP activity and peroxisomal fractions purified from P. brasiliensis yeast cells were obtained. Crude extract proteins, SDS-extracted cell wall proteins, and extracted cell wall proteins from yeast cells were subjected to SDS-PAGE analysis, blotted onto nylon membrane

and reacted to polyclonal anti-PbMLSr antibody. PbMLS was detected in crude extract (Fig.

1B, lane 3), and in SDS-extracted cell wall proteins (Fig. 1B, lane 4), but was not detected in extracted cell-wall proteins (Fig. 1B, lane 5). PbMLS activity was evaluated in SDS-extracted cell wall and in crude extract, showing specific activity of 2131.2 U/mg and 2051.28 U/mg, respectively. No cross-reactivity to the pre-immune rabbit serum was evidenced with the samples (Fig. 1C). To determined whether PbMLS was secreted to the medium, proteins were extracted from culture filtrates harvested from P. brasiliensis which had been growing for 24 and 36 h (Fig. 1D, lanes 1 and 2, respectively), 7 days (Fig. 1D, lane 3), and 14 days (Fig. 1D, lane 4). The proteins were subjected to SDS-PAGE analysis, blotted onto nylon membrane and reacted to polyclonal anti-PbMLSr antibody. PbMLS was detected in all these preparations (Fig. 1D, lanes 1 Q-VD-Oph research buy to 4). No signal was detected in medium free of cells (Fig. 1D, lane 5). PbMLS activity was evaluated in culture filtrate showing specific activity of 1305.3 U/mg. No cross-reactivity to

the pre-immune rabbit serum was evidenced with the samples (Fig. 1E). Altogether, these results suggest Dehydratase that PbMLS binds weakly to the cell wall and is actively secreted in P. brasiliensis. Since PbMLS has the AKL tripeptide, a peroxisomal/glyoxysomal signal which targets PTS1 [31], the presence of the protein was investigated in this cellular compartment. Peroxisomal and mitochondrial fractions purified of P. brasiliensis were obtained. The proteins were subjected to SDS-PAGE analysis, blotted onto nylon membrane and reacted to the polyclonal anti-PbMLSr antibody. PbMLS was detected in the peroxisomal fraction (Fig. 1F, lane 1) confirming the localization of PbMLS in this organelle. Since PbMLS has not been found in mitochondria, the mitochondrial fraction was used as the negative control (Fig. 1F, lane 2). Cellular localization of PbMLS by confocal microscopy To observe the cellular location of PbMLS, P. brasiliensis yeast cells were grown in rich medium and visualized by laser confocal microscopy. The expression of PbMLS was highly positive in the budding cells (Fig. 2 B, C and 2F) but was usually negative (Fig. 2 B and 2C) or weakly positive (Fig. 2 D) in the mother cells.