Figure 3
MPIase is essential for cell growth. (a) Accumulation of PA is relieved on Tam41p induction. The indicated cells harboring plasmids pAra-CdsA and RKP153 (for Tam41p expression from the T7 promoter), cultivated in LB medium in the presence and absence of 0.2% arabinose (for CdsA induction) or 0.1 mM IPTG (for Tam41p induction), were labeled with [14C] palmitic acid. Phospholipids were analyzed by TLC and autoradiography (top). Each reference was analyzed at the right. PE, phosphatidylethanolamine; PG, phosphatidylglycerol; CL, cardiolipin. The PA content in phospholipids was determined (bottom). (b) The pH-sensitive cdsA8 mutation was complemented by Tam41p expression. EK23 (ΔcdsA) cells transformed with either pTet-CdsA (wt) or pTet-CdsA8 (CdsA8) with (right) or without (left) Tam41p expression (by pTac-Tam41p), were streaked onto LB plates at pH 8.5. (c) PA accumulation by cdsA8 was relieved by Tam41p expression. Phospholipids in the indicated cells were analyzed by TLC and visualized with anisaldehyde/H2SO4. (d) Tam41p expression does not restore MPIase expression. The MPIase level in cells cultivated as in (a) was determined by immunoblotting. (e) MPIase is essential for cell growth. KS44 and KS46 harboring plasmids pAra-CdsA and RKP153 were streaked onto LB plates supplemented with 0.2% arabinose (for CdsA induction) or 0.1 mM IPTG (for Tam41p induction), and then incubated at 37 °C for 16 h. (f,g) M13 procoat is accumulated on MPIase depletion even in the presence of Tam41p. KS22 and KS23 cells harboring plasmids pAra-CdsA, pTet-Tam41p and pMS119-PC were subjected to pulse-chase experiments, as described in Fig. 2d. M13 procoat and coat were immunoprecipitated, followed by SDS-PAGE/autoradiography. The percentage of procoat, obtained in (f), was determined (g).
