We have identified two temperature-sensitive peroxisome-deficient mutants of Hansenula polymorpha (ts6 and ts44) within a collection of ts mutants which are impaired for growth on methanol at 43°C but grow well at 35°C. In both strains peroxisomes were completely absent in cells grown at 43°C; the major peroxisomal matrix enzymes alcohol oxidase, dihydroxyacetone synthase and catalase were synthesized normally but assembled into the active enzyme protein in the cytosol. As in wild-type cells, these enzymes were present in peroxisomes under permissive growth conditions (≤37°C). However, at intermediate temperatures (38-42°C) they were partly peroxisome-bound and partly resided in the cytosol. Genetic analysis revealed that both mutant phenotypes were due to monogenic recessive mutations mapped in the same gene, designated PER13. After a shift of per13-6ts cells from restrictive to permissive temperature, new peroxisomes were formed within 1 h. Initially one-or infrequently a few-small organelles developed which subsequently increased in size and multiplied by fission during prolonged permissive growth. Neither mature peroxisomal matrix nor membrane proteins, which were present in the cytosol prior to the temperature shift, were incorporated into the newly formed organelles. Instead, these proteins remained unaffected (and active) in the cytosol concomitant with further peroxisome development. Thus in H. polymorpha alternative mechanisms of peroxisome biogenesis may be possible in addition to multiplication by fission upon induction of the organelles by certain growth substrates.
Abstract Mitofilin proteins are crucial organizers of mitochondrial architecture. They are located in the inner mitochondrial membrane and interact with several protein complexes of the outer membrane, thereby generating contact sites between the two membrane systems of mitochondria. Within the inner membrane, mitofilins are part of hetero-oligomeric protein complexes that have been termed the mitochondrial inner membrane organizing system (MINOS). MINOS integrity is required for the maintenance of the characteristic morphology of the inner mitochondrial membrane, with an inner boundary region closely apposed to the outer membrane and cristae membranes, which form large tubular invaginations that protrude into the mitochondrial matrix and harbor the enzyme complexes of the oxidative phosphorylation machinery. MINOS deficiency comes along with a loss of crista junction structures and the detachment of cristae from the inner boundary membrane. MINOS has been conserved in evolution from unicellular eukaryotes to humans, where alterations of MINOS subunits are associated with multiple pathological conditions.
The peroxisomal flavoprotein alcohol oxidase (AO) is an octamer (600 kDa) consisting of eight identical subunits, each of which contains one flavin adenine dinucleotide molecule as a cofactor. Studies on a riboflavin (Rf) auxotrophic mutant of the yeast Hansenula polymorpha revealed that limitation of the cofactor led to drastic effects on AO import and assembly as well as peroxisome proliferation. Compared to wild-type control cells Rf-limitation led to 1) reduced levels of AO protein, 2) reduced levels of correctly assembled and activated AO inside peroxisomes, 3) a partial inhibition of peroxisomal protein import, leading to the accumulation of precursors of matrix proteins in the cytosol, and 4) a significant increase in peroxisome number. We argue that the inhibition of import may result from the saturation of a peroxisomal molecular chaperone under conditions that normal assembly of a major matrix protein inside the target organelle is prevented.
Sixteen monoclonal antibodies which recognize different cell surface antigens of the ichthyotoxic marine dinoflagellate Gyrodinium cf aureolum were prepared and characterized for use in identification by both immunofluorescence microscopy and flow cytometry.Based on the labeling results obtained from indirect immunofluorescence assays the monoclonals could be divided into 3 groups: (I) fluorescence of the overall cell surface, (11) fluorescence of the overall cell surface together with the flagella, and (111) fluorescence of granular-like structures randomly distributed along the cell surface and at the edges of the sulcus and cingulum.Cross-reactivity of 6 selected monoclonals with other phytoplankton species revealed that labeling was restricted to G. aureolum/G.cf.aureolum and to the morphologically closely related species Gymnodinium nagasakiense and G. mikimotoi.This suggests a taxonomic proximity of these species.Fixation of samples with paraformaldehyde produced the optimal immunochemical response, resulting in the strongest fluorescence intensities.In flow cytometric analyses, labeled cells of G. cf.aureolum could be dist~nguished sufficiently from unlabeled controls when monoclonals of Group I1 and a pooled serum (combined antibodies of Groups 1, 11, and 111) were used.This immunochemical technique will be applied in an early warning system to detect G. aureolum cells at dilute concentrations using flow cytometry or, alternatively, image analysis.
We have studied the role of flavin adenine dinucleotide (FAD) in the in vivo assembly of peroxisomal alcohol oxidase (AO) in the yeast Hansenula polymorpha. In previous studies, using a riboflavin (Rf) autotrophic mutant, an unequivocal judgement could not be made, since Rf-limitation led to a partial block of AO import in this mutant. This resulted in the accumulation of AO precursors in the cytosol where they remained separated from the putative peroxisomal AO assembly factors. In order to circumvent the peroxisomal membrane barrier, we have now studied AO assembly in a peroxisome-deficient/Rf-autotrophic double mutant (delta per1.rif1) of H. polymorpha. By sucrose density centrifugation and native gel electrophoresis, three conformations of AO were detected in crude extracts of delta per1.rif1 cells grown under Rf-limitation, namely active octameric AO and two inactive, monomeric forms. One of the latter forms lacked FAD; this form was barely detectable in extracts wild-type and delta per1 cells, but had accumulated in the cytosol of rif1 cells. The second form of monomeric AO contained FAD; this form was also present in delta per1 cells but absent/very low in wild-type and rif1 cells. In vivo only these FAD-containing monomers associate into the active, octameric protein. We conclude that in H. polymorpha FAD binding to the AO monomer is mediated by a yet unknown peroxisomal factor and represents the crucial and essential step to enable AO oligomerization; the actual octamerization and the eventual crystallization in peroxisomes most probably occurs spontaneously.
