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

Chimeric proteins K D K K and KPKK could not be distinguished from wild-type Kre2p, permitting the conclusion that the Kre2p TMD in the context of a native protein is not necessary for correct Golgi localization. Chimeric construct KD-K which comprises the Kre2p cytoplasmic tail, the DPAP B membrane-spanning domain, a Kre2p partial stem region, and the Kre2 protein mannosyltransferase domain was correctly localized to the Golgi complex. This indicates that the first 36 amino acid residues of the stem region of Kre2p are dispensable for Golgi targeting. The results obtained with this fusion protein again show that the Kre2p TMD is not required for Golgi retention. The fact that in the KD-K construct, the Kre2p TMD and part of the stem region are not present, argues that in the context of a protein containing a Kre2p luminal catalytic domain the cytoplasmic tail of Kre2p is sufficient to correctly target such a chimera to the Golgi complex, un- Figure I0 . Localization of chimeric proteins KKKP and KKP. Yeast cells carrying a PH08 disruption (pho8::TRP1) in a SEY6210 background and containing the different chimeric constructions on YEp352 were fixed, spheroplasted, attached to polylysine-coated glass slides, and incubated with anti-Pho8p antibodies followed by a subsequent incubation with a FITCcoupled anti-rabbit secondary Ab and DAPI. Colocalization of vacuolar localized chimeric proteins with the yeast vacuolar membrane H+-ATPase was achieved by coincubation of fixed cells along with mAb 13Dll (Kane et al., 1992) followed by incubation of treated cells with Texas red-conjugated anti-mouse secondary antibodies. Arrows point to Golgi punctiform structures.