An extracellular Lipid Transfer protein is partially bound to membranes

PAGNUSSAT L.A., REGENTE M. Y DE LA CANAL L

Iguazú

Congreso; XXXX Anual Meeting SAIB; 2004

SAIB

Lipid Transfer Proteins (LTPs) are low-molecular-mass pro-teins extensively studied in higher plants. We have previously characterized Ha-AP10, a sunflower antifungal protein homo-logous to members of the LTP family. Ha-AP10 has been de-tected in the extracellular fluids of sunflower seeds and its cDNA presents a typical secretory signal peptide sequence. Since previous experiments suggested that Ha-AP10 might be associated to membranes, the objective of this work was to de-termine if a fraction of Ha-AP10 was effectively membrane bound. To this aim, we separated membrane-associated and soluble proteins by centrifugation. The absence of cytoplasmic contamination in the membrane fraction was confirmed by tes-ting the presence of glucose-6-phosphate-dehydrogenase acti-vity. Western blot analysis revealed that Ha-AP10 is present in similar amounts both in the membrane and soluble fractions and that it is released from the membranes by non-ionic de-tergents. Bioinformatic analysis revealed that Ha-AP10 does contain neither transmembrane domains nor myristoylation signal sequences. After treatment of the membrane fraction with 0.1M Na2CO3 or 0.6M NaCl, Ha-AP10 was found in the supernatant, indicating that is loosely bound to membranes. This result was confirmed by isolating perispheric proteins by phase separation in Triton X-114. Assays are in progress to understand the biological significance of this finding.