Isolation and characterization of the plasma membrane from the yeast Pichia pastoris

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.     

A model for the tertiary structure of mammalian mitochondrial transfer RNAs lacking the entire 'dihydrouridine' loop and stem

The mammalian mitochondrial tRNA(AGY)Ser is unique in lacking the entire dihydrouridine arm. This reduces its secondary structure to a 'truncated cloverleaf'. Experimental evidence on the tertiary structure has been obtained by chemically probing the conformation of both the bovine and human species in their native conformation and at various stages of denaturation. A structural model of the bovine tRNA is presented based on the results of this chemical probing, on a comparison between nine homologous 'truncated cloverleaf' secondary structures and on analogies with the crystal structure of yeast phenylalanine tRNA. The proposed structure is very similar in shape to that of yeast tRNA(Phe) but is slightly smaller in size. It is defined by a unique set of tertiary interactions. Structural considerations suggest that other mammalian mitochondrial tRNAs have smaller dimensions as well.     

Degradation of Hydrocarbons by Members of the Genus Candida: I. Hydrocarbon Assimilation

A representative number of species of the genus Candida were tested for their ability to utilize n-alkanes of 9 to 18 carbon atoms and even-numbered 1-alkenes of 10 to 18 carbon atoms. A high percentage of these organisms exhibited the ability to assimilate some member of the hydrocarbon series employed. These findings indicate that the ability to assimilate hydrocarbons is not limited to a few species of the genus and may be of some taxonomic significance.     

Evidence for Mass Flow in Flowering Individuals of the Submersed Vascular Plant Myriophyllum heterophyllum

Myriophyllum heterophyllum Michx. is a rhizomatous submersed aquatic plant that produces a short, emergent floral spike. We hypothesized that lacunar pressures in emergent spikes should be at or near atmospheric pressure and that a mass flow of gases from submersed stems through the rhizome to emergent stems may occur as lacunar O2 concentrations and pressures in submersed stems increase during photosynthesis. We examined the potential for a pressure gradient ([delta]P) to develop along this pathway by measuring diurnal changes in lacunar gas composition and pressure in submersed stems of nonflowering plants and emergent stems of flowering individuals. Methane release from emergent spikes was also monitored during three diurnal cycles to evaluate the hypothesis that the [delta]P is maintained by the release of lacunar gases to the atmosphere. Lacunar O2 concentrations and pressures in submersed stems increased at sunrise and reached maximum levels by midday. Although O2 fluctuated similarly in emergent stems, lacunar pressures remained at or near atmospheric pressure, indicating that a [delta]P is generated between submersed and emergent stems during photosynthesis. Methane release from emergent spikes increased as lacunar pressures increased, indicating that rhizome gases are transported through emergent stems by mass flow and the [delta]P is maintained by venting lacunar gases from emergent spikes. The potential for mass flow in both flowering and nonflowering individuals is discussed.     

Identification and sequence analysis of genes involved in late steps in cobalamin (vitamin B12) synthesis in Rhodobacter capsulatus.

A 6.4-kb region of a 6.8-kb BamHI fragment carrying Rhodobacter capsulatus genes involved in late steps of cobalamin synthesis has been sequenced. The nucleotide sequence and genetic analysis revealed that this fragment contains eight genes arranged in at least three operons. Five of these eight genes show homology to genes involved in the cobalamin synthesis of Pseudomonas denitrificans and Salmonella typhimurium. The arrangement of these homologous genes differs considerably in the three genera. Upstream of five overlapping genes (named bluFEDCB), a promoter activity could be detected by using lacZ fusions. This promoter shows no regulation by oxygen, vitamin B12 (cobalamin), or cobinamide. Disruption of the bluE gene by a Tn5 insertion (strain AH2) results in reduced expression of the puf and puc operons, which encode pigment-binding proteins of the photosynthetic apparatus. The mutant strain AH2 can be corrected to a wild-type-like phenotype by addition of vitamin B12 or cobinamide dicyanide. Disruption of the bluB gene by an interposon (strain BB1) also disturbs the formation of the photosynthetic apparatus. The mutation of strain BB1 can be corrected by vitamin B12 but not by cobinamide. We propose that a lack of cobalamin results in deregulation and a decreased formation of the photosynthetic apparatus.     

Glucose Metabolism in Sediments of a Eutrophic Lake: Tracer Analysis of Uptake and Product Formation

The uptake of glucose and the formation of end products from glucose catabolism have been measured for sediments of eutrophic Wintergreen Lake with a combination of tritiated and ¹⁴C-labeled tracers. Time course analyses of the loss of [³H]glucose from sediments were used to establish rate constants for glucose uptake at natural substrate concentrations. Turnover times from these analyses were about 1 min for littoral and profundal sediments. No seasonal or site differences were noted in turnover times. Time course analyses of [U-¹⁴C]glucose uptake and ¹⁴C-labeled end product formation indicated that glucose mass flow could not be calculated from end product formation since the specific activity of added [¹⁴C]glucose was significantly diluted by pools of intracellular glucose and glucose metabolites. Mass flow could only be accurately estimated by use of rates of uptake from tracer studies. Intermediate fermentation end products included acetate (71%), propionate (15%), lactate (9%), and only minor amounts of butyrates or valerates. Addition of H₂ to sediments resulted in greater production of lactate (28%) and decreased formation of acetate (50%), but did not affect glucose turnover. Depth profiles of glucose uptake indicated that rates of uptake decreased with depth over the 0- to 18-cm interval and that glucose uptake accounted for 30 to 40% of methanogenesis in profundal sediments.