An amphitropic cAMP-binding protein in yeast mitochondria. 1. Synergistic control of the intramitochondrial location by calcium and phospholipid

Abstract

A CAMP-binding protein is found to be integrated into the inner mitochondrial membrane of the yeast Saccharomyces cereuisiae under normal conditions. It resists solubilization by high salt and chaotropic agents. The protein is, however, converted to a soluble form which then resides in the intermembrane space, when isolated mitochondria are incubated with low concentrations of calcium. Phospholipids or diacylglycerol (or analogues) dramatically increases the efficiency of receptor release from the inner membrane, whereas these compounds alone are ineffective. Also, cAMP does not effect or enhance liberation from the membrane of the CAMP-binding protein. Photoaffinity labeling with 8-N3-[32P]cAMP followed by mitochondrial subfractionation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis does not reveal differences in the apparent molecular weight between the membrane-bound and the soluble form of the c A M P receptor. The two forms differ, however, in their partitioning behavior in Triton X-114 as well as in their protease resistance, indicating that the release from the membrane is accompanied by a change in lipophilicity and conformation of the receptor protein. Evidence is presented that a change of the intramitochondrial location of the yeast CAMP-binding protein also occurs in vivo and leads to the activation of a mitochondrial CAMP-dependent protein kinase. The CAMP-binding protein is the first example of a mitochondrial protein with amphitropic character; Le., it has the property to occur in two different locations, as a membrane-embedded and a soluble form.;Several proteins, previously described as soluble cytoplasmic components, such as a-actinin, gelsolin, profilin, spectrin, vinculin, and protein kinase C have been found in recent years to occur in a membrane-associated form as well [e.g., Niggli and Burger (1987); for a review, see Burn (1988a)l. The reversible interaction of these proteins with the plasma membrane plays an important role in the fulfillment of their function in proliferation, the cell cycle, cell division and motility [for a review, see Geiger (1 985) and Burridge (1 986)]. It is controlled by the turnover of certain phospholipids in the membrane (Burn & Burger, 1987;Burn, 1988b). For instance, upon hydrolytic generation of diacylglycerol in the plasma membrane the soluble Ca2+-and phosphatidylserine-dependent protein kinase C becomes associated with the cytoplasmic side of the plasma membrane, with a concomitant decrease of the Ca2+ requirement for its activation [Kishimoto et a]., 1980; for a review, see Nishizuka (1986)l.On the other hand, a variety of enzymes have been described, the greatest portion of which is tightly anchored in the membrane from which they can be released, most commonly, by a chemical modification or hydrolytic cleavage step. In this group are found, e.g., CTP:phosphocholine cytidylyltransferase (Kraft & Anderson, 1983), calmodulin-dependent protein kinase type I1 (Bennet et al., 1983), and the selfprocessing protease calpain (Imajoh et...

The sulphonylurea drug, glimepiride, stimulates release of glycosylphosphatidylinositol-anchored plasma-membrane proteins from 3T3 adipocytes

“…Adenylate cyclase was measured as described by Salomon et al (1974). Lactate dehydrogenase was assayed by a standard spectrophotometric assay (Wroblewski and LaDue, 1955 Rodel et al (1985) and Muller and Bandlow (1989) KCI,5mM MgCl2, 1 mM MnCl2, 0.5 mM EDTA, 0.2mM DTT, 1 mM IBMX, 0.1 mM PMSF, 0.1 mM AMP, 0.2% octyl glucoside). About 100 jtg of protein was incubated with 100 nCi [3H]cAMP (about 2 nmol) in the presence or absence of 0.8 mM cAMP in a total volume of 250,ul for 5 min at 4 'C.…”

Glucose-Induced Sequential Processing of a Glycosyl-Phosphatidylinositol-Anchored Ectoprotein in Saccharomyces cerevisiae

“…Cells from S. cerevisiae ABYS-1 were grown in semisynthetic lactate medium as described previously (34) to 5 ϫ 10 6 cells per ml at 30ЊC, washed two times in 0.1 M Tris-SO 4 (pH 8.5)-1.1 M sorbitol, then resuspended in amino acid-depleted or inositol-free semisynthetic lactate medium supplemented with 0.05% yeast extract at the same titer, and grown to 10 7 cells per ml. The cells were centrifuged, resuspended in the above-described medium lacking yeast extract at 7.5 ϫ 10 7 cells per ml, and incubated (2 h, 30ЊC).…”

Strain-dependent Occurrence of Functional GTP:AMP Phosphotransferase (AK3) in Saccharomyces cerevisiae

“…Spheroplasts were prepared from midlogarithmic cultures (1-2 ϫ 10 7 cells per ml) and lysed in 0.45 M mannitol. Mitochondria were prepared from the 10,000 ϫ g pellet and purified by Percoll gradient centrifugation as described previously (Mü ller and Bandlow, 1989).…”

“…This assumption was also based on results obtained with a Pak3/mouse dihydrofolate reductase fusion protein that was found to be located exclusively in mitochondria (Schricker et al, 1992a). To determine the subcellular location of yeast Aky3p unambiguosly, we subfractionated cells and mitochondria (Rödel et al, 1985;Mü ller and Bandlow, 1989) from strain D273-10B as well as from AKY3 multi-copy transformants. The success of the subfractionation was controlled immunologically by Western blotting using antibodies directed against specific topological marker proteins (Fig.…”