The lipidome and proteome of microsomes from the methylotrophic yeast Pichia pastoris

• A method for isolation of ER fractions (microsomes) from P. pastoris was developed.
• Lipidome and proteome analysis of P. pastoris microsomes (M30 and M40) was performed.
• 30,000 g and 40,000 g microsomes differ in the degree of fatty acid saturation.
• Microsomes contain ceramides, hexoxyl ceramides and inositol containing sphingolipids.
• 294 microsomal proteins were identified, 20 of them not assigned to a function.

The methylotrophic yeast Pichia pastoris is a popular yeast expression system for the production of heterologous proteins in biotechnology. Interestingly, cell organelles which play an important role in this process have so far been insufficiently investigated. For this reason, we started a systematic approach to isolate and characterize organelles from P. pastoris. In this study, we present a procedure to isolate microsomal membranes at high purity. These samples represent endoplasmic reticulum (ER) fractions which were subjected to molecular analysis of lipids and proteins. Organelle lipidomics included a detailed analysis of glycerophospholipids, fatty acids, sterols and sphingolipids. The microsomal proteome analyzed by mass spectrometry identified typical proteins of the ER known from other cell types, especially Saccharomyces cerevisiae, but also a number of unassigned gene products. The lipidome and proteome analysis of P. pastoris microsomes are prerequisite for a better understanding of functions of this organelle and for modifying this compartment for biotechnological applications.