Exploring Functions on Yarrowia lipolytica Sterol Carrier Protein 2 (YLSCP-2) in Yeast

GIANOTTI, ALEJO R.; BRUM, LUCIANO A.; ERMÁCORA, MARIO R.; FERREYRA, RAUL G.

Rosario

Congreso; IX Congreso Argentino de Microbiología General SAMIGE 2013; 2013

Sociedad Argentina de Microbiología General SAMIGE

The ascomycetous yeast Yarrowia lipolytica is currently used as a model for the study of protein secretion, peroxisome biogenesis, dimorphism, hydrophobic substrates utilization, and exploited in several application 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. SCP-2 coding sequences are present as protein domains or individual genes, in genomes from all the superkingdoms of life. We have previously shown that Y. lipolytica SCP-2 gene product (YLSCP-2) is a 128-amino-acid basic protein inducible by fatty acids (1) and it is located in the yeast peroxisomes(2). YLSCP2 is able to bind a variety of lipids and transfer them to phospholipid membranes by a collision-mediated mechanism.2 Its structure was recently resolved in our lab (PDB code 4JGX)(3). Like in Y. lipolytica, transgenic expression of YLSCP-2 in S. cereviciae analyzed by sub-cellular fractionation, showed preferential localization of the protein to peroxisomes. Induction of catalase marker activity was also seen. In order to confirm those findings, GFP fusions to YLSCP-2 were constructed and immunofluorescence imaging analysis was performed. GFP fusions were done leaving ?free? the C-terminal ?NNL? peroxisomal targeting sequence (PTS) of YLSCP-2, in order to allow organellar import. In addition, ELISA and western-blot analysis of cytoplasm and peroxisome enriched fractions of the yeast cells mechanically broken also confirm import of GFP-YLSCP-2 fusions to the matrix of peroxisomes. Another genetic approach we are using to evaluate YLSCP-2 functions in yeast is the generation of Y. lipolytica null mutants of the gene. The physiological consequences of YLSCP-2 absence were assessed by nutritional and biochemical studies. Null mutant growth on and utilization of alkanes, fatty acids, ethanol, and acetate as a sole source of carbon are presented, and the requirement for full YLSCP-2 function in yeast under nutritional challenges will be discuss. (1)Ferreyra et al. 2006. Arch. Biochem. Biophys. 453(2): 197-206. (2)Falomir et al.2009. Biophys. J. 97(1): 248-56. (3)Perez De Berti et al. Manuscript in prep.