식물 세포 내 수분 통로 단백질의 조절 기작 및 중합체 형성 기작에 관한 연구

Abstract

The water channel aquaporin that transports water across lipid membranes is widely distributed in all types of organisms and also at multiple types of cellular membranes in a given cell. The biological function and detailed structure of aquaporin have been well understood. Especially for plants, a fine tuned adaptation of physiology including the water balance appears to be of crucial importance. An adjustment of the respective physiological process could be achieved by regulation mechanisms, which range from post-translational modification, molecular trafficking and to tetramer formation of aquaporin. In this study, I investigated the regulation mechanisms of plasma membrane intrinsic protein2 (PIP2) protein, a major group of plant AQP proteins by two independent approaches. First, as one of the post-translational modification, I investigated whether ubiquitination is involved in PIP2 regulation under abiotic responses in higher plants. Rma1H1, a hot pepper (Capsicum annuum L.) homolog of a human RING membrane-anchor 1 E3 ubiquitin ligase, was rapidly induced by various abiotic stresses including dehydration, and its overexpression in transgenic Arabidopsis plants conferred strongly enhanced tolerance to drought stress. Colocalization experiments with marker proteins revealed that Rma1H1 resides in the ER membrane. Overexpression of Rma1H1 in Arabidopsis inhibited trafficking of an aquaporin isoform PIP2

1 from the ER to the plasma membrane and reduced PIP2

1 levels in protoplasts and transgenic plants. This Rma1H1-induced reduction of PIP2

1 was inhibited by MG132, an inhibitor of the 26S proteasome. Furthermore, Rma1H1 interacted with PIP2

1 in vitro and ubiquitinated it in vivo. Similar to Rma1H1, Rma1, an Arabidopsis homolog of Rma1H1, showed same aspects in the plant cell. Based on these results, I propose that Rma1H1 and Rma1 play a critical role in the down regulation of plasma membrane aquaporin levels by inhibiting aquaporin trafficking to the plasma membrane and subsequent proteasomal degradation as a response to dehydration in transgenic Arabidopsis plants.Secondly, I focused on the mechanism of the tetramer formation of aquaporins. I investigated the sequence critical for tetramer formation of Arabidopsis plasma membrane aquaporin AtPIP2

1 by amino acid substitution mutagenesis. Alanine substitution mutation at the C-terminal 10-amino acid (aa) segment of TMD1 to TMD5, but not TMD6, prevented the tetramer formation but instead caused AtPIP2

1 proteins to further polymerize into high molecular weight forms. In addition, alanine substitution mutation at either C- or N-terminal 10-aa segment of TMD2, TMD4 and TMD5 significantly weakened the interaction between AtPIP2

1 monomers. AtPIP2

1[TMD2-A2] and AtPIP2

1[TMD5-A1] that underwent higher levels of polymerization accumulated in the ER to high levels, in addition to a portion of them being targeting to the plasma membranes, when expressed as GFP fusion proteins in transgenic plants. Furthermore, in contrast to AtPIP2

1:GFP and AtPIP2

1[TMD2-A3]:GFP that conferred enhanced sensitivity to dehydration stress, AtPIP2

1[TMD2-A2]:GFP and AtPIP2

1[TMD5-A1]:GFP caused an enhanced resistance to dehydration stress. Based on these results, I propose that both the type and location of individual amino acid residues in the TMDs play critical roles in the tetramer formation and the interaction between monomers.