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

The extent of colocalization of Kre2p with Mnnlp or Kexlp was scored by quantitating in a given dual localization experiment 30 cells that contained clearly defined signals representing 200-250 individual fluorescent punctate spots for each antigen. Compiled data revealed that Mnnlp gave rise to approximately the same number of punctate spots per cell as Kre2p and 75% of the punctiform fluorescent spots from Texas red and FITC overlapped. Kexlp gave rise to ~10% fewer punctate spots per cell compared with Kre2p-associated signals, and 65% of the punctiform fluorescent spots emanating from both proteins did not overlap. Finally, the intracellular localization of chimeric protein KKP was quantitatively scored by examining 750 individual ceils containing clearly defined signals. Punctiform fluorescence different from nucleus, ER, or vacuole was defined as Golgi localization. Vacuolar localization was determined by colocalization with the 60-kD vacuolar membrane H+-ATPase subunit.