Functional Expression of Yarrowia lipolytica Sterol Carrier Protein 2 (YLSCP-2) in Saccharomyces cereviciae

FERREYRA, RAUL G.; GIANOTTI, ALEJO; ERMÁCORA, MARIO R.

Mar del Plata, Buenos Aires

Congreso; VIII Congreso Argentino de Microbiología General SAMIGE 2012; 2012

Sociedad Argentina de Microbiología General SAMIGE

Yarrowia lipolytica is one of the most extensively studied ‘‘non-conventional’’ yeasts which is currently used as a model for the study of protein secretion, peroxisome biogenesis, dimorphism, degradation of hydrophobic substrates, and several new fields. The entire sequence of the six Y. lipolytica chromosomes has been determined. The sterol carrier protein-2 (SCP-2) is a nonspecific lipid transfer protein that has been implicated in the transfer and metabolism of cholesterol, branched-chain fatty acids, acyl-CoA conjugates, and other lipids. Its gene was found in genomes from the three superkingdoms of life. We have previously shown that Y. lipolytica SCP-2 (YLSCP-2), a 128-amino-acid protein inducible by fatty acids1, is located in peroxisomes of the yeast and binds a variety of lipids which can be transferred from YLSCP2 to phospholipid membranes by a collision-mediated mechanism2. Taking into account that Saccharomyces cereviciae has no SCP-2 gene and its metabolism has difficulty to adapt growing on lipids, the physiological consequences of YLSCP-2 transgenic expression in the yeast were analyzed. To evaluate recombinant YLSCP-2 function in vivo, intracellular distribution of the protein in S. cereviciae complemented cells was assessed and nutritional and biochemical studies were performed. Growth and utilization of palmitic acid, ethanol, and sodium acetate as a sole source of carbon, were followed for parental and recombinant S. cereviciae strains, showing slightly differences. However, peroxisomal marker catalase activity was found to be increased for recombinant S. cereviciae expressing YLSCP-2, in all nutritional conditions. To measure the intracellular concentration of YLSCP-2, an ELISA was developed and applied to the analysis of the cytoplasm and peroxisome enriched fractions of the yeast cells mechanically broken. It was found that almost all the protein targeted to the organellar fraction. These results confirmed that transgenic expression in S. cereviciae of YLSCP-2, like in Y. lipolytica, is also preferentially located in the peroxisomal fraction of yeast cell lysate. Moreover, catalase induction in the recombinant strain showed an apparent expansion of the peroxisomal compartment. Even so, the inferred concentration of YLSCP-2 in peroxisomes and the protein affinity for long-chain fatty acids are high enough, like in Y. lipolytica1, to make YLSCP-2 to function as a lipid carrier inside the organelle of complemented cells of S. cereviciae. Data presented here is being confronted to another genetic approach in the lab: Y. lipolytica null mutants for the YLSCP-2 gene are being generated to evaluate the physiological consequences of its absence. 1Ferreyra et al. 2006. Arch. Biochem. Biophys. 453(2): 197-206. 2Falomir et al.2009. Biophys. J. 97(1): 248-56.