Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative ␤-1-4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also upregulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H2O2. These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H2O2 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.cellulose ͉ fenton ͉ lignin ͉ cellulase ͉ brown-rot

The vacuole/lysosome serves an essential role in allowing cellular components to be degraded and recycled under starvation conditions. Vacuolar hydrolases are key proteins in this process. In Saccharyomces cerevisiae, some resident vacuolar hydrolases are delivered by the cytoplasm to vacuole targeting (Cvt) pathway, which shares mechanistic features with autophagy. Autophagy is a degradative pathway that is used to degrade and recycle cellular components under starvation conditions. Both the Cvt pathway and autophagy employ double-membrane cytosolic vesicles to deliver cargo to the vacuole. As a result, these pathways share a common terminal step, the degradation of subvacuolar vesicles. We have identified a protein, Cvt17, which is essential for this membrane lytic event. Cvt17 is a membrane glycoprotein that contains a motif conserved in esterases and lipases. The active-site serine of this motif is required for subvacuolar vesicle lysis. This is the first characterization of a putative lipase implicated in vacuolar function in yeast.One fundamental role of the yeast vacuole is in the recycling of biological macromolecules. The vacuole, like the lysosome in animal cells, is the primary site of degradation. Our understanding of the hydrolytic vacuolar enzymes that serve in protein turnover is well advanced. Progress has also been made in elucidating mechanisms that deliver the substrates of these vacuolar hydrolases. While research has focused on the biosynthesis and function of the vacuolar proteases, little is known about how lipids are recycled in this organelle, and a lipase that functions in membrane recycling has not been identified.Nearly all vacuolar/lysosomal delivery pathways involve packaging of cargo within membrane-enclosed transport compartments. Because the vacuole/lysosome serves as the final destination for these numerous vesicle-mediated transport pathways, the issue of how membranes reaching the vacuole are recycled is an important one. Macroautophagy is the major degradative process in eukaryotes and is essential during starvation conditions (1). In yeast, autophagy overlaps with a biosynthetic process, the Cvt pathway, that delivers the hydrolase aminopeptidase I (API 1 ; Ref.2) from its site of synthesis in the cytoplasm to the vacuolar lumen. Cvt and autophagy employ many of the same molecular components and are mechanistically related (3-6). Both pathways involve the formation of double-membrane cytosolic vesicles, sequestering either precursor aminopeptidase I (prAPI) specifically, or in the case of autophagy, also enveloping bulk cytosol in a nonselective manner. Fusion of these vesicles with the vacuole results in the release of single-membrane subvacuolar vesicles within the lumen. These pathways require a mechanism for specific lysis of the internalized vesicles, so that vesicle cargo can be released into the vacuole lumen, and further require a mechanism for degradation of vesicle lipids.To understand the molecular basis of these import and degradation pathways, we carried out a g...

A role for DnaK, the major E. coli Hsp70, in chaperoning de novo protein folding has remained elusive. Here we show that under nonstress conditions DnaK transiently associates with a wide variety of nascent and newly synthesized polypeptides, with a preference for chains larger than 30 kDa. Deletion of the nonessential gene encoding trigger factor, a ribosome-associated chaperone, results in a doubling of the fraction of nascent polypeptides interacting with DnaK. Combined deletion of the trigger factor and DnaK genes is lethal under normal growth conditions. These findings indicate important, partially overlapping functions of DnaK and trigger factor in de novo protein folding and explain why the loss of either chaperone can be tolerated by E. coli.

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