Fig. 4. OGT is associated with the mitochondrial inner membrane. (A) Immunogold staining of purified mitochondria demonstrates an inner membrane localization for OGT; no specific label was detected when the gold-labeled secondary antibody was used alone (negative control). Bars, 0.1 µM. (B) Purified HeLa mitochondria (whole) were fractionated by sonication and high-speed centrifugation into membrane and soluble protein fractions, separated on a 4-20% PAGE-gel, transferred to nitrocellulose and probed with OGT-specific, affinity-purified antibodies. Equal amounts of protein were loaded in each lane. The majority of mOGT was found associated with the membrane fraction after salt washing (see Materials and Methods). Fractionation of mitochondria was monitored by mtHSP 70 content. The bulk of this matrix marker appeared in the soluble fraction under conditions where mOGT remained membrane associated. (C) Localization of mOGT was compared in mitochondria and mitoplasts subjected to alkaline extraction with carbonate buffer. The outer membrane of mitochondria was removed by hypotonic lysis to produce the mitoplasts. Both mitochondria and the mitoplasts were washed with 100 mM sodium carbonate pH 11 to remove peripherally associated proteins. The soluble and membrane fractions were separated and probed for OGT. The arrows indicate that the mOGT is greatly enriched in the membrane fractions of both mitochondria and mitoplasts.