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20EGY2 is a zinc -containing, intramembrane protease, located in the thylakoid membrane. It 21 is consider to be involved in the regulated intramembrane proteolysis -a mechanism leading

Aquaporins (AQPs), also known as water channel proteins, are members of a large protein family termed Major Intrinsic Proteins (MIP). The mammalian AQPs have been most comprehensively described, while knowledge about AQPs in invertebrates is limited mainly to insects. Not a single AQP protein has been described in snails to date. Consequently, we decided to search for the proteins in gastropod representatives, namely Lymnaea stagnalis, Catascopia occulta, and Stagnicola palustris (Mollusca; Gastropoda; Pulmonata; Lymnaeidae). Using the molecular approach, we identified L. stagnalis, C. occulta,and S. palustris open reading frames (ORFs) showing homology to AQP genes available in GenBank database, and characterized the encoded proteins, referred to as LsAQP1, CoAQP1, and SpAQP1, respectively. The putative snail aquaporins contain 299 amino acids, have a molecular mass of about 32 kDa, display the general AQP topology and three-dimensional structure congruent with orthodox AQPs, i.e., water-specific ones. Due to high levels of similarity in their characteristics, LsAQP1 was chosen for further studies, as the obtained results were supposed to be applicable for CoAQP1 and SpAQP1. Expression analysis revealed the presence of LsAQP1 transcript in the digestive tract, the cerebral ganglia, the kidney, the reproductive system, and the foot, suggesting that LsAQP1 as well as CoAQP1 and SpAQP1 are ubiquitous proteins and may play important roles in many essential water transport processes. The role appears to be confirmed by results of the yeast growth complementation assay pointing at functionality of LsAQP1. Thus, the obtained results support the AQP expression in gastropod tissues for the first time.Electronic supplementary materialThe online version of this article (doi:10.1007/s00232-014-9629-0) contains supplementary material, which is available to authorized users.

EGY2 is a zinccontaining, intramembrane protease, located in the thylakoid membrane. It is consider to be involved in the regulated intramembrane proteolysis -a mechanism leading to activation of membrane-anchored transcription factors through proteolytic cleavage, which causes them to be released from the membrane. The physiological functions of EGY2 in chloroplasts remains poorly understood. To answer the question what is the significance of EGY2 in chloroplast functioning two T-DNA insertion lines devoid of EGY2 protein were obtained and the mutants phenotype and photosystem II parameters were analyzed.Chlorophyll fluorescence measurements revealed that the lack of EGY2 protease caused changes in non-photochemical quenching (NPQ) and minimum fluorescence yield (F 0 ) as well as higher sensitivity of photosystem II (PSII) to photoinhibition. Further immunoblot analysis revealed significant changes in the accumulation levels of the three chloroplast-encoded PSII core apoproteins: PsbA (D1) and PsbD (D2) forming the PSII reaction centre and PsbCa protein component of CP43, a part of inner PSII antennae. The accumulation level of nuclearencoded proteins Lhcb1-3 -a components of the major light-harvesting complex II (LHCII) as well as proteins forming minor peripheral antennae complexes, namely Lhcb4 (CP29), Lhcb5 (CP26), and Lhcb6 (CP24) remain, however, unchanged. The lack of EGY2 led to a significant increase in the level of PsbA (D1) with simultaneous decrease in accumulation levels of PsbC (CP43) and PsbD (D2). To test the hypothesis that the observed changes in the abundance of chloroplast-encoded proteins are a consequence of changes in gene expression levels, real-time PCR was performed. The obtained results shown that egy2 mutants display an increased expression of PSBA and reduction in the PSBD and PSBC genes. Simultaneously pTAC10, pTAC16 and FLN1 proteins were found to accumulate in thylakoid membranes of analyzed mutant lines. These proteins interact with core complex of plastid encoded RNA polymerase and may be involved in the regulation of chloroplast gene expression.