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

To analyze further the targeting role of the three nonenzymatic domains of Kre2p and to remove the possible additional complexity of a targeting mechanism involving the mannosyltransferase portion of Kre2p, combinations of noncatalytic domains of Kre2p were tested to assess what region of Kre2p was sufficient to target a Pho8p reporter protein to the Golgi complex. As opposed to the results obtained with fusion protein KD-K, all three noncatalytic domains of the Kre2 protein were found to be required for full Golgi retention of the Pho8p luminal region (KKKP). These results are in agreement with those of Chapman and Munro (1994) who found that a fusion protein containing the NHz terminus, the membrane-spanning domain, and a partial stem region of Kre2p linked to a reporter protein was retained in the Golgi complex. The mostly vacuolar localization of KKP demonstrated that the first part of the Kre2p stem region which is not required for retention in the context of a Kre2p catalytic domain (KD-K), is necessary, in combination with the Kre2p cytoplasmic tail and TMD for the targeting of the Pho8p catalytic portion to the Golgi complex (KKKP). Taken together, the intracellular localizations of chimeric proteins KD-K, KKP, and KKKP suggest that the Kre2p luminal domain does play a role in Golgi localization.