Deletion of the PalB MIT domain impairs its association with membranes
and the processing of PacC. A, MIT domain-mediated association of
PalB with membrane fractions. Wild-type palB800 and mutant
palB801ΔMIT protoplast lysates
were fractionated into 13,000 × g insoluble (P13) and 100,000
× g insoluble (P100) and soluble (S100) fractions, which were
analyzed by Western blotting. B,
palB801ΔMIT results in weak
loss-of-function in diagnostic plate tests of pH signaling
(16,
74). palB800 is
phenotypically wild-type (data not shown). In contrast,
palB801ΔMIT confers some
neomycin resistance (Neo), weakly impairs growth on alkaline pH (OH)
and molybdate (Mo) plates, and decreases tolerance of LiCl
(Li), indicating that deletion of the MIT domain results in weak
loss-of-function; SC is synthetic complete medium without any addition.
C, similar stability of PalB and PalBΔMIT in pH
shift experiments. PalB was detected using α-HA antibody. Similar
loading in the different lanes was determined using β-actin.
palB800 and palB801ΔMIT
cells cultured at acidic ambient pH were shifted to alkaline pH. Cell extracts
were analyzed by Western blotting. D,
palB801ΔMIT impairs PacC processing
in pH shift experiments. Wild-type palB800 and null
palBΔ controls are shown. The three forms of PacC are
indicated.