The methylotrophic yeast Hansenula polymorpha is a recognised model system for investigation of peroxisomal function, special metabolic pathways like methanol metabolism, of nitrate assimilation or thermostability. Strain RB11, an odc1 derivative of the particular H. polymorpha isolate CBS4732 (synonymous to ATCC34438, NRRL-Y-5445, CCY38-22-2) has been developed as a platform for heterologous gene expression. The scientific and industrial significance of this organism is now being met by the characterisation of its entire genome. The H. polymorpha RB11 genome consists of approximately 9.5 Mb and is organised as six chromosomes ranging in size from 0.9 to 2.2 Mb. Over 90% of the genome was sequenced with concomitant high accuracy and assembled into 48 contigs organised on eight scaffolds (supercontigs). After manual annotation 4767 out of 5933 open reading frames (ORFs) with significant homologies to a non-redundant protein database were predicted. The remaining 1166 ORFs showed no significant similarity to known proteins. The number of ORFs is comparable to that of other sequenced budding yeasts of similar genome size.

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MDY2, a gene required for efficient mating of the yeast Saccharomyces cerevisiae, was characterized in this study. The gene encodes a protein of 212 amino acids, which contains a ubiquitin-like (UBL) domain (residues 74-149). Deletion of MDY2 is associated with a five- to sevenfold reduction in mating efficiency, mainly due to defects in nuclear migration and karyogamy at the prezygotic stage. However, prior to mating pair fusion, shmoo formation is reduced by 30%, with a concomitant failure to form mating pairs. Strikingly, migration of the nucleus into the shmoo tip is also delayed or fails to occur. In addition, we show that in mdy2 mutants, microtubule bundles, as well as the microtubule end-binding protein Kar9, fail to localize properly to the shmoo tip, suggesting that the nuclear migration defect could be due to aberrant localization of Kar9. Pheromone signal transduction (as measured by FUS1 induction by α-factor) is not affected in mdy2Δ mutants and mitosis is also normal in these cells. MDY2 is not induced by mating pheromone. In vegetatively growing cells, GFP-Mdy2 is localized in the nucleus, and remains nuclear after exposure of cells to α-factor. His-tagged Mdy2 shows no evidence of the C-terminal processing typical of ubiquitin, and also localizes to the nucleus. Thus MDY2 is a novel gene, whose product plays a role in shmoo formation and in nuclear migration in the pre-zygote, possibly by interacting with other UBL-type proteins that possess ubiquitin association (UBA) domains.

The gene encoding for bacterio-opsin (bop gene) from Halobacterium halobium has been introduced in a yeast expression vector. After transformation in Schizosaccharomyces pombe, bacterio-opsin (BO) is expressed and was detected by antisera. The precursor protein of BO (pre-BO) is processed by cleavage of amino acids at the N-terminal end as in H. halobium. Addition of the chromophore, retinal, to the culture medium results in a slight purple colour of the yeast cells indicating the in vivo regeneration of BO to bacteriorhodopsin (BR) and its incorporation into membranes. Therefore, in contrast to the expression in E. coil, isolation of the membrane protein and reconstitution in lipid vesicles is not necessary for functional analysis. The kinetics of the ground state signal of the photocycle BR in protoplasm is demonstrated by flash spectroscopy and is comparable to that of the natural system. The present investigation shows for the first time the transfer of an energy converting protein from archaebacteria to eukaryotes by genetic techniques. This is a basis for further studies on membrane biogenesis, genetics, and bioenergetics by analysis of in vivo active mutants.

MDY2 encodes a ubiquitin-like (UBL)-domain protein necessary for efficient mating in Saccharomyces cerevisiae. Unlike most UBL proteins, Mdy2 is apparently not subject to C-terminal processing and is localized predominantly in the nucleus. Deletion of MDY2 is associated with a five- to seven-fold reduction in mating efficiency, mainly due to defects in nuclear migration and karyogamy at the prezygotic stage. Here, we looked for two potential interacting partners of Mdy2, investigated the function of Mdy2 in nuclear movement, determined the increased heat sensitivity defects of mdy2Δ mutants, and inspected localization of Mdy2. Coprecipitation studies show that Mdy2 associates with α-tubulin and with the microtubule (MT)-associated dynactin subunit p150(Glued)/Nip100. nip100Δ mutants exhibit no defects in nuclear migration or in MT length or orientation during shmooing growth. Deletion of MDY2 display small nuclear migration phenotype during vegetative growth and seems to exacerbate the defects in mitotic nuclear migration seen in the nip100Δ strain. Deletion of MDY2 increased heat sensitivity of the cells and these strains accumulate mitotic nuclear migration defects and shortened MTs under these conditions. GFP-Mdy2 proteins which are localized predominantly in the nucleus at permissive temperature are localized to cytoplasmic foci during heat shock. Colocalization studies revealed that heat stress-induced enrichment of Mdy2 in cytoplasmic foci merged mainly with stress granules marker Pab1. During glucose deprivation a minority of Mdy2 foci overlapped with P-bodies marker Dcp2, while most Mdy2 foci and Pab1 foci overlap. Accordingly, we propose that Mdy2 plays a critical role in the MT-dependent processes of karyogamy and stress response.