The recruitment of specific cytosolic proteins to intracellular membranes through binding phosphorylated derivatives of phosphatidylinositol (PtdIns) controls such processes as endocytosis, regulated exocytosis, cytoskeletal organization, and cell signaling. Protein modules such as FVYE domains and PH domains that bind specifically to PtdIns 3-phosphate (PtdIns-3-P) and polyphosphoinositides, respectively, can direct such membrane targeting. Here we show that two representative Phox homology (PX) domains selectively bind to specific phosphatidylinositol phosphates. The PX domain of Vam7p selectively binds PtdIns-3-P, while the PX domain of the CPK PI-3 kinase selectively binds PtdIns-4,5-P(2). In contrast, the PX domain of Vps5p displays no binding to any PtdInsPs that were tested. In addition, the double mutant (Y42A/L48Q) of the PX domain of Vam7p, reported to cause vacuolar trafficking defects in yeast, has a dramatically decreased level of binding to PtdIns-3-P. These data reveal that the membrane targeting function of the Vam7p PX domain is based on its ability to associate with PtdIns-3-P, analogous to the function of FYVE domains.

SUMMARYThree forms of disulfide-linked insulin receptor complexes are labeled by covalent cross-linking to receptor-bound 12S l-insulin in native adipocyte or liver membranes. These receptors of M r 350,000, M r 320,000, and M r 290,000 are composed of a(M r 125,000), /3(M r 90,000), and /3,(M r 49,000) subunits in stoichiometries of (a/3) 2 , {ap)(ap,), and (afi,) 2 , respectively. In adipocyte membranes, these receptor structures can undergo a first step of reduction by dithiothreitol, dissociating into M r 210,000 (a/3) and M r 160,000 (a/3,) partially reduced receptor fragments. Complete dissociation of such fragments into the free a, /3, and /S, receptor subunits is achieved at high reductant concentrations. In liver plasma membranes the partially reduced receptor species of M r 210,000 and M r 160,000 are observed even when electrophoresis is performed under nonreducing conditions. This observation indicates that native liver plasma membranes contain multiple redox states of the high affinity insulin receptor. DIABETES 29:945-947, November 1980. P revious reports from this laboratory 1 " 5 have demonstrated that insulin receptors in several tissues of the rat and human can be specifically labeled by covalent cross-linking of receptor-bound 125 l-insulin with disucciminidyl suberate and other related compounds. Dodecyl sulfate polyacrylamide gel electrophoresis, followed by autoradiographic analysis of intact rat fat cell insulin receptors labeled using this technique, showed that they exist as disulfide-linked complexes with M r 350,000." Dissociation of the M r 350,000 insulin receptor complex by dithiothreitol treatment before electrophoresis showed that it consists of 2 subunit types, the a subunit (M r 125,000) and the p subunit (M r 90.000).

Acetyl coenzyme A carboxylase and fatty acid synthetase activities were studied to determine the biochemical basis of the markedly impaired capacity of fat cells from spontaneously obese, old rats to convert glucose to fatty acids relative to cells from lean, young rats. Michaelis constants for the substrates of both enzymes were similar in large and small adipocyte homogenates. In contrast, Vmax values were over 80% less in homogenates from large relative to small cells on a per cell basis. Long-term dialysis or the presence of albumin during the assays failed to restore the activities of these enzymes in homogenates of large fat cells. The combination of equal volumes of homogenates from the two cell types resulted in carboxylase and synthetase activities intermediate between activities found in the two homogenates alone. Therefore, the presence of endogenous allosteric inhibitors does not appear to account for the markedly blunted fatty acid synthesis enzyme activities in large fat cells. These results suggest that the fatty acid synthesis impairment, which is a primary defect in the insulin resistance of the large cells, is at least partly due to diminished cellular contents of acetyl coenzyme A carboxylase and fatty acid synthetase.