Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to a medial-Golgi compartment

In parallel with the localization studies, all chimeric proteins were assessed functionally. They were verified to be membrane associated and properly glycosylated (data not shown), showing that they possess a type II membrane orientation (see Fig. 6 ). In addition, their in vitro and in vivo enzymatic activities were assayed since low levels of mannosyltransferase activity would imply abnormal secondary structure of a particular chimera and possible mislocaliza-tion in the secretory pathway. The in vitro cd,2-mannosyltransferase activity was assayed in cell-membrane fractions. The specific activity of each chimeric protein is displayed in Fig. 9 , where KRE2 expressed in a kre2A strain was arbitrarily determined to be 100%, a value just slightly lower than that of wild-type mannosyltransferase activity from a genomic copy of KRE2. KDKK, KPKK, DKKK, as well as MR/KKK appear to be fully active. KD-K displays intermediate levels of mannosyltransferase activity suggesting that the stem region from position 31 to 66 is necessary for folding or proper catalytic activity.