Isolation of hepatic mitochondrial contact sites: Previously unrecognized inner membrane components

An improved, fast, and relatively simple procedure for isolation of hepatic mitochondrial contact sites is described. These contact sites include conventional outer membrane, but the inner membrane component (which we term fusion patches) has a unique biochemical composition characterized by a clustering of three specific inner membrane proteins of 54,52, and 31 kDa identified by proteomics, respectively, as the alpha and beta subunits of ATP synthase and the liver isoform of adenine nueleotide transferase. The contact site fraction was prepared using a discontinuous sucrose gradient from crude outer membranes derived from swollen/shrunk rat liver mitochondria. The resultant contact sites were analyzed using a continuous sucrose density gradient, revealing an apparent heterogeneity due to varying amounts of retained fusion patches in relation to the unvarying outer membrane component. By electron microscopy, contact sites consist of small vacuoles that contain one or several tiny vesicles, many of which are composed of multiple, closely packed lamellae. The contact site subfraction morphology is consistent with the biochemical variation. Thus, contact sites are not haphazard fusions of outer and inner membrane, but consist in part of regions of inner membrane of novel composition (fusion patches) and of conventional outer membrane.