PURIFIED VESICLES OF TOBACCO CELL VACUOLAR AND PLASMA-MEMBRANES EXHIBIT DRAMATICALLY DIFFERENT WATER PERMEABILITY AND WATER CHANNEL ACTIVITY

The vacuolar membrane or tonoplast (TP) and the plasma membrane (PM) o f tobacco suspension cells were purified by free-flow electrophoresis (FFE) and aqueous two-phase partitioning, with enrichment factors from a crude microsomal fraction of greater than or equal to 4- to 5-fold and reduced contamination by other cellular membranes, For each purifi ed fraction, the mean apparent diameter of membrane vesicles was deter mined by freeze-fracture electron microscopy, and the osmotic shrinkin g kinetics of the vesicles were characterized by stopped-flow light sc attering. Osmotic water permeability coefficients (P-f) of 6.1 +/- 0.2 and 7.6 +/- 0.9 mu m.s(-1) were deduced for PM-enriched vesicles puri fied by FFE and phase partitioning, respectively, The associated activ ation energies (E-a; 13.7 +/- 1.0 and 13.4 +/- 1.4 kcal.mol(-1), respe ctively) suggest that water transport in the purified PM occurs mostly by diffusion across the lipid matrix, In contrast, water transport in TP vesicles purified by FFE was characterized by (i) a 100-fold highe r P-f of 690 +/- 35 mu m.s(-1), (ii) a reduced E-a of 2.5 +/- 1.3 kcal .mol(-1), and (iii) a reversible inhibition by mercuric chloride, up t o 83% at 1 mM, These results provide functional evidence for channel-m ediated water transport in the TP, and more generally in a higher plan t membrane. A high TP P-f suggests a role for the vacuole in buffering osmotic fluctuations occurring in the cytoplasm, Thus, the differenti al water permeabilities and water channel activities observed in the t obacco TP and PM point to an original osmoregulatory function for wate r channels in relation to the typical compartmentation of plant cells.