Role of C-terminal Domain and Transmembrane Helices 5 and 6 in Function and Quaternary Structure of Major Intrinsic Proteins

Abstract

We previously observed that aquaporins and glycerol facilitators exhibit different oligomeric states when studied by sedimentation on density gradients following nondenaturing detergent solubilization. To determine the domains of major intrinsic protein (MIP) family proteins involved in oligomerization, we constructed protein chimeras corresponding to the aquaporin AQPcic substituted in the loop E (including the proximal part of transmembrane domain (TM) 5) and/or the C-terminal part (including the distal part of TM 6) by the equivalent domain of the glycerol channel aquaglyceroporin (GlpF) (chimeras called AGA, AAG, and AGG). The analogous chimeras of GlpF were also constructed (chimeras GAG, GGA, and GAA). cRNA corresponding to all constructs were injected into Xenopus oocytes. AQPcic, GlpF, AAG, AGG, and GAG were targeted to plasma membranes. Water or glycerol membrane permeability measurements demonstrated that only the AAG chimera exhibited a channel function corresponding to water transport. Analysis of all proteins expressed either in oocytes or in yeast by velocity sedimentation on sucrose gradients following solubilization by 2% n-octyl glucoside indicated that only AQPcic and AAG exist in tetrameric forms. GlpF, GAG, and GAA sediment in a monomeric form, whereas GGA and AGG were found mono/dimeric. These data bring new evidence that, within the MIP family, aquaporins and GlpFs behave differently toward nondenaturing detergents. We demonstrate that the Cterminal part of AQPcic, including the distal half of TM 6, can be substituted by the equivalent domain of GlpF (AAG chimera) without modifying the transport specificity. Our results also suggest that interactions of TM 5 of one monomer with TM 1 of the adjacent monomer are crucial for aquaporin tetramer stability.

Developmental Regulation of the Direct Interaction between the Intracellular Loop of Connexin 45.6 and the C Terminus of Major Intrinsic Protein (Aquaporin-0)

“…Alternatively it has been hypothesized that MIP(AQP0) plays other unidentified functions in lens. Our study reported here implies that this interaction appears not to be related to the water transporting activity of MIP(AQP0) because Duchesne et al (36) report that the C terminus of MIP(AQP0), the Cx45.6 binding domain, does not interfere with the transport activity of the protein. Studies conducted by Dunia et al (37) and by our laboratory (18) demonstrate that the localization of MIP(AQP0) is closely correlated with newly formed gap junctional plaques at the narrow zone of the lens bow area where young fiber cells are actively differentiating, implying that the interaction between MIP(AQP0) and Cx45.6 may facilitate the assembly of the Cx45.6 into nascent gap junction plaques.…”

Interactions between Plasma Membrane Aquaporins Modulate Their Water Channel Activity

“…The replacement of loop E in a "weak" aquaporin, AQP0, with loop E from a more active aquaporin, AQP2, increases the P f of oocytes, and this correlates with an increase in the amount of the protein in the plasma membrane (Kuwahara et al, 1999). Another study showed that exchanging loop E of GlpF and AQPcic modified channel solute specificity and targeting to the plasma membrane (Duchesne et al, 2002). The effect of single, double, or triple mutations in ZmPIP1;1 loop E on P f induction when coexpressed with ZmPIP2;5 should reveal the roles of specific residues.…”

“…Amino acid sequence comparison of ZmPIP1;1 and ZmPIP1;2 revealed significant amino acid residue substitutions in loop E, in which V246, Q250, H251, and A255 of ZmPIP1;1 are replaced, respectively, by I247, R250, D251, and N256 of ZmPIP1;2 ( Figure 8A). The important role of loop E in AQP function and in the interaction between monomers in the tetramer has been reported (Jung et al, 1994;Duchesne et al, 2002). To investigate whether loop E of ZmPIP1;2 was involved in its function and its interaction with another PIP, a chimeric protein, ZmPIP1;1LE, consisting of ZmPIP1;1 in which loop E was replaced with loop E from ZmPIP1;2 (amino acid residues 247 to 256), was constructed.…”

Stability of the Glycerol Facilitator in Detergent Solutions

“…In all previous reports of detergent solutions of the glycerol facilitator, the protein appeared predominantly monomeric by several assays, although SDS-PAGE sometimes showed varying amounts of dimer, trimer, tetramer, and highermolecular weight species (27,(38)(39)(40)(41)(42)(43)(44). These observations have led to the suggestion that, in contrast to the aquaporins, the glycerol facilitators exist as monomers or weakly associating tetramers in membranes.…”

“…The paucity of membrane protein structures has precluded systematic investigations of their quaternary structures, but it is worth noting that bacteriorhodopsin crystallizes both as a monomer (35) and as a trimer (36,37). The oligomeric state of the GF has been the subject of numerous investigations by sedimentation on sucrose density gradients (38)(39)(40), cryo-electron microscopy (41,42), freeze-fracture electron microscopy (43,44), chemical cross-linking (27), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (27,(38)(39)(40)(41)(42)(43)(44). Most investigators have found the protein to exist as a monomer in SDS and as a mixture of oligomers in octyl glucoside, N-lauryl sarcosine, Triton X-100, and yeast, E. coli, and Xenopus membranes.…”