Hansenula polymorpha Swi1p and Snf2p are essential for methanol utilisation

We have cloned the Hansenula polymorpha SWI1 and SNF2 genes by functional complementation of mutants that are defective in methanol utilisation. These genes encode proteins similar to Saccharomyces cerevisiae Swi1p and Snf2p, which are subunits of the SWI/SNF complex. This complex belongs to the family of nucleosome-remodeling complexes that play a role in transcriptional control of gene expression. Analysis of the phenotypes of constructed H. polymorpha SWI1 and SNF2 disruption strains indicated that these genes are not necessary for growth of cells on glucose, sucrose, or various organic nitrogen sources which involve the activity of peroxisomal oxidases. Both disruption strains showed a moderate growth defect on glycerol and ethanol, but were fully blocked in methanol utilisation. In methanol-induced cells of both disruption strains, two peroxisomal enzymes involved in methanol metabolism, alcohol oxidase and dihydroxyacetone synthase, were hardly detectable, whereas in wild-type cells these proteins were present at very high levels. We show that the reduction in alcohol oxidase protein levels in H. polymorpha SWI1 and SNF2 disruption strains is due to strongly reduced expression of the alcohol oxidase gene. The level of Pex5p, the receptor involved in import of alcohol oxidase and dihydroxyacetone synthase into peroxisomes, was also reduced in both disruption strains compared to that in wild-type cells.

Layered control architectures in robots and vertebrates

We revieiv recent research in robotics, neuroscience, evolutionary neurobiology, and ethology with the aim of highlighting some points of agreement and convergence. Specifically, we com pare Brooks' (1986) subsumption architecture for robot control with research in neuroscience demonstrating layered control systems in vertebrate brains, and with research in ethology that emphasizes the decomposition of control into multiple, intertwined behavior systems. From this perspective we then describe interesting parallels between the subsumption architecture and the natural layered behavior system that determines defense reactions in the rat. We then consider the action selection problem for robots and vertebrates and argue that, in addition to subsumption- like conflict resolution mechanisms, the vertebrate nervous system employs specialized selection mechanisms located in a group of central brain structures termed the basal ganglia. We suggest that similar specialized switching mechanisms might be employed in layered robot control archi tectures to provide effective and flexible action selection

Deviant Pex3p levels affect normal peroxisome formation in Hansenula polymorpha:A sharp increase of the protein level induces the proliferation of numerous, small protein-import competent peroxisomes

Pex3p has been implicated in the biosynthesis of the peroxisomal membrane of the yeast Hansenula polymorpha. Here we show that in the initial stages of a sharp increase in Pex3p levels, induced in batch cultures of cells of a constructed H. polymorpha strain, which contained seven copies of PEX3 under control of the alcohol oxidase promoter (WT::P-AOX.PEX3(7x)), strongly interfered with normal peroxisome proliferation. Ultrastructural studies demonstrated that in such cells numerous small peroxisomes had developed, which were absent in wild-type controls. These organelles, which contained typical peroxisomal matrix and membrane proteins (alcohol oxidase, catalase, Pex3p, Pex10p and Pex14p), showed a relatively low density(1.18 g cm(-3)) after sucrose gradient centrifugation of WT::P-AOX.PEX3(7x) homogenates, compared to normal peroxisomes (1.23 g cm(-3)). We furthermore demonstrated that these early induced, small peroxisomes were protected against glucose-induced proteolytic degradation and did not fuse to form larger organelles. Remarkably, the induction of these small peroxisomes was paralleled by a partial defect in matrix protein import, reflected by the mislocalization of minor amounts of alcohol oxidase protein in the cytosol. However, when the cells were subsequently placed under conditions in which the synthesis of a new matrix enzyme (amine oxidase) was induced while simultaneously the excessive proliferation was repressed (by repression of the P-AOX), amine oxidase protein was selectively incorporated into these organelles. This indicated that the small peroxisomes had regained a normal protein import capacity. Based on these results we argue that peroxisome proliferation and matrix protein import are coupled processes in H. polymorpha. (C) 1997 John Wiley &amp; Sons, Ltd.</p

Deviant Pex3p levels affect normal peroxisome formation in Hansenula polymorpha: A sharp increase of the protein level induces the proliferation of numerous, small protein-import competent peroxisomes

Pex3p has been implicated in the biosynthesis of the peroxisomal membrane of the yeast Hansenula polymorpha. Here we show that in the initial stages of a sharp increase in Pex3p levels, induced in batch cultures of cells of a constructed H. polymorpha strain, which contained seven copies of PEX3 under control of the alcohol oxidase promoter (WT::P-AOX.PEX3(7x)), strongly interfered with normal peroxisome proliferation. Ultrastructural studies demonstrated that in such cells numerous small peroxisomes had developed, which were absent in wild-type controls. These organelles, which contained typical peroxisomal matrix and membrane proteins (alcohol oxidase, catalase, Pex3p, Pex10p and Pex14p), showed a relatively low density(1.18 g cm(-3)) after sucrose gradient centrifugation of WT::P-AOX.PEX3(7x) homogenates, compared to normal peroxisomes (1.23 g cm(-3)). We furthermore demonstrated that these early induced, small peroxisomes were protected against glucose-induced proteolytic degradation and did not fuse to form larger organelles. Remarkably, the induction of these small peroxisomes was paralleled by a partial defect in matrix protein import, reflected by the mislocalization of minor amounts of alcohol oxidase protein in the cytosol. However, when the cells were subsequently placed under conditions in which the synthesis of a new matrix enzyme (amine oxidase) was induced while simultaneously the excessive proliferation was repressed (by repression of the P-AOX), amine oxidase protein was selectively incorporated into these organelles. This indicated that the small peroxisomes had regained a normal protein import capacity. Based on these results we argue that peroxisome proliferation and matrix protein import are coupled processes in H. polymorpha. (C) 1997 John Wiley & Sons, Ltd