D.M. Roberts, N. Chanmanivone
Dept of Biochem. Cell, and Molec. Biol., Univ. of Tennessee, Knoxville, TN 37996, USA
Nodulin 26 is a member of the Major Intrinsic Protein (MIP) superfamily of membrane channel proteins that includes water and solute transporters. The protein is expressed in nitrogen-fixing nodules on soybean roots and is targeted to the symbiosome membrane that encloses the endosymbiotic rhizobia bacterium within infected cells of the nodule. It constitutes the predominant protein component of this membrane (Weaver et al. 1991). In previous work we have shown that soybean nodulin 26 is an aquaglyceroporin that mediates the flux of water and uncharged solutes across the symbiosome membrane (Dean et al. 1999). However, compared to most aquaporins, nodulin 26 shows a substantially lower rate of water transport. For example, compared to the prototypical aquaporin, mammalian AQP 1, nodulin 26 has a 30- to 50-fold lower unitary conductance (Dean et al. 1999). An examination of various aquaporins shows that the rate and selectivity of transport can be modulated by a variety of exogenous factors including pH and phosphorylation (Nemeth-Cahalan, Hall 2000; Zeuthen, Klaerke 1999; Yasui et al. 1999). To address the factors that regulate the permeability of nodulin 26, we have investigated the effects of
pH, Ca and phosphorylation on the regulation of nodulin 26 transport.
The transport properties of nodulin 26 were evaluated upon expression in Xenopus oocytes as described previously (Dean et al. 1999). The intrinsic transport rate of nodulin 26 was elevated three-fold by either lowering the pH in the recording bath or by phosphorylation of serine 262 within the carboxyl-terminal region of the protein. In contrast, it was found that nodulin 26 transport was substantially inhibited (5- to 6-fold) by the elevation of intracellular calcium within the oocyte.
Overall, the results suggest that the aquaporin activity of nodulin 26 is modulated by pH and calcium, factors that may impact water flow and osmoregulation of the symbiosome membrane. With respect to pH effects, the regulation of nodulin 26 aquaglyceroporin activity may be coupled to the electrogenic proton pump that acidifies the lumen of the symbiosome, particularly under conditions of high metabolic activity (Uvardi, Day 1997). The effects of calcium are more complex. In vivo, nodulin 26 is phosphorylated on a unique serine residue (S262 within the carboxyl terminal domain) by a calcium-dependent protein kinase that is co-localized on the symbiosome membrane (Weaver et al. 1991). The present results suggest that this event would enhance the transport rate of the protein. However, assay in Xenopus oocytes also suggests that Ca2+ ion alone inhibits the activity, perhaps through a separate mechanism (e.g. calmodulin binding as has been reported for other MIP proteins; Nemeth-Cahalan, Hall 2000). The opposing effects of calcium and the possible interplay of phosphorylation in regulating these effects, as well as the larger question of the symbiotic role of up and down regulation of nodulin 26 transport, needs to be investigated further. (Supported by USDA NRICGP 9703548 and NSF MCB-9904978.)
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