Adenylate cyclase in Saccharomyces cerevisiae is a peripheral membrane protein.

Abstract

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains the CYR] polypeptide, responsible for catalyzing formation of cyclic AMP (cAMP) from ATP, and two RAS polypeptides, which mediate stimulation of cAMP synthesis of guanine nucleotides. By analogy to the mammalian enzyme, models of yeast adenylate cyclase have depicted the enzyme as a membrane protein. We have concluded that adenylate cyclase is only peripherally bound to the yeast membrane, based on the following criteria: (i) substantial activity was found in cytoplasmic fractions; (ii) activity was released from membranes by the addition of 0.5 M NaCI; (iii) in the presence of 0.5 M NaCI, activity in detergent extracts had hydrodynamic properties identical to those of cytosolic or NaCI-extracted enzyme; (iv) antibodies to yeast adenylate cyclase identified a full-length adenylate cyclase in both membrane and cytosol fractions; and (v) activity from both cytosolic fractions and NaCl extracts could be functionally reconstituted into membranes lacking adenylate cyclase activity. The binding of adenylate cyclase to the membrane may have regulatory significance; the fraction of activity associated with the membrane increased as cultures approached stationary phase. In addition, binding of adenylate cyclase to membranes appeared to be inhibited by cAMP. These results indicate the existence of a protein anchoring adenylate cyclase to the membrane. The identity of this protein remailns unknown.

The 70-kilodalton adenylyl cyclase-associated protein is not essential for interaction of Saccharomyces cerevisiae adenylyl cyclase with RAS proteins.

“…As shown in Fig. 1B, a major band of the CYR1 protein gave a molecular size of about 200 to 220 kDa on an SDS-polyacrylamide gel, as reported previously (12,17,27,34,43), and no apparent degradation was observed. The amount of the CYR1 protein in each fraction roughly corresponded to the intensity of its Mn2+-dependent adenylyl cyclase activity.…”

“…The apparent molecular weights were estimated as about 890,000 for the RAS-responsive adenylyl cyclase and about 670,000 for the Mn2+-dependent protein. The sizes were much larger than the observed molecular size (-220 kDa) for the CYR1 product monomer (12,27,34,43), indicating formation of complexes. The complexes were resistant to treatment with 0.04% SDS in addition to 1% Lubrol PX-0.5 M NaCl, and inclusion of protease inhibitors (leupeptin at 10 p,M, pepstatin at 1 p,M, and bestatin at 1 p,M) in the cell lysis and extraction buffer did not affect the elution pattern (data not shown).…”

Activation of adenylate cyclase in cdc25 mutants of Saccharomyces cerevisiae

“…From these findings a biochemical link has been suggested between Cdc25 and adenylate cyclase in the absence of Ras. Moreover, it has been suggested that the binding of adenylate cyclase to the membrane might have regulatory significance [25]. We have now determined the distribution of adenylate cyclase in cells carrying both RAS gene products, in the presence and in the absence of Cdc25.…”

Large-scale analysis of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae.

“…The Cap/ Srv2 gene product stimulates adenylate cyclase (FedorChaiken et al 1990;Field et al 1990). Adenylate cyclase and its activators, Cdc25 and Ras, appear to reside at the plasma membrane (Willumsen et al 1986;Deschenes and Broach 1987;Mitts et al 1990).…”