Phosphoglucomutase in Saccharomyces cerevisiae is a cytoplasmic glycoprotein and the acceptor for a Glc-phosphotransferase.

UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor in vertebrates and Paramecium tetraurelia is a cytoplasmic 62-kDa glycoprotein. To determine if the yeast Saccharomyces cerevisiae also possesses Glc-phosphotransferase activity, a crude cellular lysate was incubated with [beta-32P]UDP-Glc and analyzed. A phosphoglycoprotein having an apparent molecular mass of 62 kDa (pgp62) was found to be the predominant labeled macromolecule. Reconstitution experiments determined that both a soluble and membrane fraction were required for labeling, and suggested that the Glc-phosphotransferase is membrane-associated while pgp62 is cytoplasmic. The reaction is evolutionarily conserved to the extent that rat liver Glc-phosphotransferase was capable of recognizing the yeast acceptor and vice versa. The yeast 62-kDa acceptor was purified, and partial amino acid sequences showed a high level of identity with rabbit muscle phosphoglucomutase. Subsequently, both yeast and rabbit muscle phosphoglucomutase were found to be acceptors in the Glc-phosphotransferase reaction. The label was found on a tryptic peptide distinct from that containing the enzyme's active site serine. When phosphoglucomutase was overexpressed, an increase was seen in Glc-phosphotransferase acceptor activity and in specific metabolic labeling of the acceptor by glucose and mannose.

Receptor synthesis and routing to the plasma membrane.

Hormones and other extracellular proteins exert their effects on the cells that they influence by interacting with receptors in the plasma membranes of these target cells. For these interactions to occur, the receptor proteins must be efficiently synthesized, processed, and transported to the plasma membrane. This review summarizes the events and organelles involved in this process.

Calmodulin and protein kinase C antagonists also inhibit the Ca2+-dependent protein protease, calpain I.

The calmodulin and C-kinase antagonists melittin, calmidazolium, N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W7), and trifluoperazine (TFP) also inhibit the activity of the human erythrocyte Ca2+-dependent protease, calpain I. W-5, the nonchlorinated derivative of W-7, was ineffective as an inhibitor of calpain I just as it is for calmodulin and protein kinase C. Dose response studies provided the following IC50 values: melittin, 2.6 microM; calmidazolium, 6.2 microM; trifluoperazine, 130 microM; W-7, 251 microM. These IC50 values indicate that the compounds have affinities 10 to 600 fold less for calpain I than for calmodulin; however, the affinities of the inhibitory compounds are comparable for calpain I and protein kinase C. Kinetic analysis indicates that the compounds are competitive inhibitors of calpain I with respect to substrate.

Activation of a calmodulin-dependent phosphatase by a Ca2+-dependent protease.

A calmodulin-dependent protein phosphatase (calcineurin) was converted to an active, calmodulin-independent form by a Ca2+-dependent protease (calpain I). Proteolysis could be blocked by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, leupeptin, or N-ethylmaleimide, but other protease inhibitors such as phenylmethanesulfonyl fluoride, aprotinin, benzamidine, diisopropyl fluorophosphate, and trypsin inhibitor were ineffective. Phosphatase proteolyzed in the absence of calmodulin was insensitive to Ca2+ or Ca2+/calmodulin; the activity of the proteolyzed enzyme was greater than the Ca2+/calmodulin-stimulated activity of the unproteolyzed enzyme. Proteolysis of the phosphatase in the presence of calmodulin proceeded at a more rapid rate than in its absence, and the proteolyzed enzyme retained a small degree of sensitivity to Ca2+/calmodulin, being further stimulated some 15-20%. Proteolytic stimulation of phosphatase activity was accompanied by degradation of the 60-kilodalton (kDa) subunit; the 19-kDa subunit was not degraded. In the absence of calmodulin, the 60-kDa subunit was sequentially degraded to 58- and 45-kDa fragments; the 45-kDa fragment was incapable of binding 125I-calmodulin. In the presence of calmodulin, the 60-kDa subunit was proteolyzed to fragments of 58, 55 (2), and 48 kDa, all of which retained some ability to bind calmodulin. These data, coupled with our previous report that the human platelet calmodulin-binding proteins undergo Ca2+-dependent proteolysis upon platelet activation [Wallace, R. W., Tallant, E. A., & McManus, M. C. (1987) Biochemistry 26, 2766-2773], suggest that the Ca2+-dependent protease may have a role in the platelet as an irreversible activator of certain Ca2+/calmodulin-dependent reactions.