The protein kinase C (encoded by PKC1 gene) is a Ser/Thr protein kinase apparently involved in the control of many cellular systems in yeast cells: the cell wall integrity pathway, the synthesis of ribosomes and the appropriated reallocation of transcription factors under specific stress conditions. In our laboratory, we have studied the involvement of protein kinase C in the control of different sugar-induced effects. Previous data from our lab indicates the involvement of Pkc1p in the activation, by glucose, of the plasma membrane ATPase (Souza, M. A. A. et al; Microbiology 147, 2849-2855; 2001). The plasma membrane H⁺-ATPase (PMA1) of Saccharomyces cerevisiae regulates intracellular pH and provides the driving force for nutrient uptake. It is a 98-kDa polypeptide, anchored in the membrane by 10 hydrophobic-helices and belonging to a widespread family of cation transporters known as the P2-type ATPases. Glucose metabolism induces a moderate and slow increase in PMA1 gene expression and triggers a rapid and strong increase in ATPase catalytic activity. The glucose activation of the yeast H⁺-ATPase results from a combined effect on the kinetic parameters, including a Km decrease and a Vmax increase. In Saccharomyces cerevisiae mutations in the regulatory domain located at the carboxyl-terminus affecting Ser 899 or Thr 912, abolish the glucose-dependent Km or Vmax change respectively (F. Portillo et al, JBC 269, 10393-10399; 1994). Moreover, a pseudosubstrate peptide (House, C. and Kemp, B. E., Science 238, 1726-1728, 1987), corresponding to positions 394-409 in the predicted Pkc1 p amino acid sequence, functions as a positive control for Pkc1 p activity in the in vitro studies. In this work we evaluate the in vitro phosphorylation of the H⁺-ATPase by Pkc1p. The plasma membrane ATPase was purified according to Becher dos Passos et al, BBA 1136, 57-67, 1992. The PKC1 gene was cloned within the vector PYES2/CT and purified using the Ni-NTA agarose resin (Qiagen). The cloned PKC1 gene was expressed in the mutant pkc1∆ to test the in vivo functionality of the expressed protein, by observing the reversion of the mutant phenotype.