INVOLVEMENT OF MITOCHONDRIAL CONTACT SITES IN THE SUBCELLULAR COMPARTMENTALIZATION OF PHOSPHOLIPID BIOSYNTHETIC-ENZYMES

The concerted synthesis of phospholipids derived from serine involving two microsomal enzymes (phosphatidylserine synthase and phosphatidyle thanolamine N-methyltransferase) and a mitochondrial one (phosphatidyl serine decarboxylase) occurs in reconstituted cell-free systems. Subfr actionation of crude mitochondria after swelling and separating on a s ucrose density gradient resulted in the isolation of two contact site- enriched fractions from total outer membranes and inner membranes, res pectively. Estimation of marker enzyme activities shows a high recover y of glucose-6-phosphate phosphatase (a marker for the endoplasmic ret iculum) associated with contact site-enriched fractions. Accordingly, the linked synthesis of phosphatidylserine, phosphatidylethanolamine, and at a lesser extent phosphatidylcholine can occur. This biosyntheti c pathway was absent from purified contact site-enriched fractions cor relative with the absence of glucose-6-phosphate phosphatase activity. Reconstitution experiments, including contact site-enriched fractions incubated with endoplasmic reticulum-rich fraction, led to the restor ation of the linked synthesis of phospholipids, thereby demonstrating that a reversible association between these two fractions can occur. T hese functional interactions between the endoplasmic reticulum and mit ochondria are confirmed at the ultra-structural level using either che mical or physical fixation before resin embedding. These results show that the interorganelle trafficking of lipids may involve only highly specialized microdomains of both membranes, thereby allowing the maint enance of a specific lipid composition and distribution within membran es.