In the present study we demonstrate that Ca 2ϩ uptake by the mitochondria suppresses the positive feedback effects of Ca 2ϩ on the IP 3 R in permeabilized hepatocytes. Moreover, our data demonstrate that the mitochondrial modulation of IP 3 -induced Ca 2ϩ release is limited to those elements of the ER Ca 2ϩ stores in proximity with the mitochondria, giving rise to subcellular heterogeneity in IP 3 sensitivity and IP 3 R excitability. These properties allow the mitochondria to play a key role in orchestrating the subcellular pattern of [Ca 2ϩ ] c signaling. EXPERIMENTAL PROCEDURESHepatocytes plated on polylysine-coated coverslips were maintained in primary culture for 18 -24 h (15, 26). Cytosolic [Ca 2ϩ ] waves in fura2-loaded intact hepatocytes were measured essentially as described previously (15,27). The cells were stimulated with vasopressin (2-20 nM) prior to and after addition of mitochondrial inhibitors or solvent in sequential runs, and the rate of wave propagation was determined in each condition (15,27). For permeabilized cell experiments, cells were loaded with fluorescent dyes (obtained from Molecular Probes or Teflabs) by incubation for 30 -60 min at 37°C in medium composed of 121 mM NaCl, 5 mM NaHCO 3 , 10 mM Na-HEPES, 4.7 mM KCl, 1.2 mM KH 2 PO 4 , 1.2 mM Mg 2 SO 4 , 2 mM CaCl 2 , 10 mM glucose, and 2% bovine serum albumin, pH 7.4, essentially as described previously (9,15,26). Dye concentrations were: 150 nM MitoTracker Green, 2 M rhod2/AM, 5 M fura2FF/AM, and 5 M fluo3FF/AM. We have shown previously that compartmentalization of rhod2 occurs in the mitochondria (15) and fura2FF is trapped in the ER (26) of hepatocytes using this loading protocol. Dye-loaded cells were washed with Ca 2ϩ -free buffer and then permeabilized by incubation for 6 min with 15 g/ml digitonin in intracellular medium (ICM) composed of 120 mM KCl, 10 mM NaCl, 1 * This work was supported by Grants DK38422 (to A. P. T.) and DK51526 (to G. H.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Various molecular forms of chromium have been implicated in the regulation of glucose metabolism, and chromium deficiency can be associated with insulin resistance and impaired glucose tolerance. Protein-tyrosine phosphatases (PTPases), which negatively regulate signaling through the insulin receptor, are potential targets of chromium action, since this transition metal may inhibit catalysis at the thiol-dependent active sites of these enzymes. Treatment of cultured rat hepatoma cells with 0.1 mM CrCl 3 for 16 h increased the insulin-stimulated tyrosine phosphorylation of high M r insulin receptor substrate (IRS) proteins by 49% to 7.3-fold over basal (n ‫ס‬ 7; P ‫ס‬ 0.03), without altering basal insulin receptor or IRS tyrosine phosphorylation or insulin-stimulated receptor autophosphorylation, suggesting a post-receptor effect of chromium on signal transduction. PTPase activity in cell extracts of CrCl 3 -treated hepatoma cells before or after insulin stimulation was unchanged, indicating that if chromium acted via cellular PTPases, the effect was reversible and limited to the in vivo state. Chromium (Cr +3 ) ion and two organic derivatives, an oligopeptide chromium complex from bovine liver (Cr-pep), and a synthetic multinuclear complex of chromium with carboxylate ligands (Sm-Cr) were also tested for their direct in vitro inhibition of the enzymatic activity of LAR and PTP1B, two structurally variant PTPases that have been implicated in regulation of the insulin signaling pathway. PTP1B (rat and human) was strongly inhibited by CrCl 3 to 21-33% of control (n ‫ס‬ 4-6; P < 0.001). In contrast, LAR activity was actually enhanced by CrCl 3 to 47% above the control value (n ‫ס‬ 12; P < 0.001). The Cr-pep and Sm-Cr complexes had no effect on PTP1B and LAR activity at the tested concentrations using the pNPP assay. These data suggest that the metabolic effects of chromium may be mediated by inhibition of PTP1B, a PTPase that negatively modulates insulin signaling, consistent with other recent studies implicating PTP1B in the regulation of the dephosphorylation of post-insulin receptor substrate proteins.

The genomes of a wide variety of organisms have now been sequenced; a major challenge ahead is to understand the function, regulation and modification of the many encoded gene products. We have been carrying out proteomics approaches to the identification and analysis of signalling pathways in yeast. 121 of 122 protein kinases were cloned and purifed from yeast as GST fusions and analyzed for their ability to phosphorylate 60 different yeast substrates. More than 93% of the kinases exhibited activities that are 5 fold or higher, relative to controls, including 18 of 24 previously uncharacterized kinases. Many protein kinases had novel activities; for example 27 yeast kinases were found to phosphorylate Tyr. In addition, we have now cloned 6000 open reading frames and overexpressed their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with a variety of different proteins, nucleic acids and phospholipids. As examples, we have probed yeast proteome chips with calmodulin and six different phospholipids. These studies revealed many new calmodulin and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. They can also be used to screen protein-drug interactions and to detect posttranslational modifications.