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

Abstract

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.

Increased dosage of AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris

“…Total fatty acid composition of the strains was further analysed to study the effect of desaturase regulation on the fatty acid profile (Supplementary Table S9). The results confirmed that the most abundant fatty acids in P. pastoris were oleic acid, linoleic acid, palmitic acid, linolenic acid and stearic acid, as it has been previously reported7071. Interestingly, it seems that in producing strains the most abundant fatty acid was linoleic acid, unlike the 0C reference strain, in which oleic acid was the main fatty acid.…”

An enhanced plant lipidomics method based on multiplexed liquid chromatography–mass spectrometry reveals additional insights into cold‐ and drought‐induced membrane remodeling

“…For lipid extraction 100‐mg aliquots from the frozen powder were immersed in a hot solvent mixture containing 60% propan‐2‐ol and further processed as described in Grillitsch et al . ().…”

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

“…However, it is currently unknown how cellulose biosynthesis is initiated and in particular whether a specific primer is required to form the first acceptor of glycosyl transfer. Several proposed models for bacterial and plant cellulose biosynthesis include the involvement of lipid-linked oligosaccharides or glycolipids, such as sitosterol-glucoside, which has been found only in plants so far (34)(35)(36). However, the catalytic activity of Pichia-expressed PttCesA8 suggests that the enzyme either initiates cellulose biosynthesis in vivo using an expression host-provided factor [as observed for Rhodobacter BcsA upon expression in E. coli (14)] or that cellulose biosynthesis initiates in vitro upon incubation with UDP-Glc and Mn 2+ .…”