The proteolytic system of the yeast Kluyveromyces lactis

Major proteolytic activities were characterized in the yeast K. lactis NRRL 1118, grown in chemostat cultures. This yeast expressed proteolytic activi ties similar to those found in S. cerevisiae. This fact was particularly ev ident in the case of proteases such as PrA, PrB and CpY with regard to subs trate specificity, activation at pH 5.0 and inhibition patterns. The presen ce of a CpS activity could not be detected in either fresh or activated cel l-free extracts by using the dipeptide N-Cbz-Gly-Leu, even in the presence of Zn+2. On the other hand, K. lactis exhibits at least two major intracell ular Ap activities different from those reported in other yeasts, and these seem to be carried out by closely related proteins. These activities corre sponded to molecular masses of about 60 kDa, close pi values, and a similar behaviour in non-denaturing polyacrylamide electrophoresis. Both activitie s were enhanced by Co2+ and inhibited by EDTA. Among different aminoacyl-p- NAs, they preferentially hydrolysed Lys-p-NA. No increase of Ap activity wa s obtained by incubation of extracts at acid pH. The maximum PrA and PrB ac tivities detected in N-limited cultures were six-fold higher than those exp ressed under C- or P-limitation. The effect of culture conditions on the Cp and Ap expression was much less pronounced in comparison with PrA and PrB activities, Ap levels even being slightly higher in C-limited cells. This f act suggests that hydrolysis of protein to peptides might be the limiting s tep in the pathway of general protein degradation in the vacuole. Copyright (C) 1999 John Wiley & Sons, Ltd.