Abul Kalam Azad

Characterization of Protein Phosphatase 2A Acting on Phosphorylated Plasma Membrane Aquaporin of Tulip Petals

A protein phosphatase holo-type enzyme (38, 65, and 75 kDa) preparation and a free catalytic subunit (38 kDa) purified from tulip petals were characterized as protein phosphatase 2A (PP2A) by immunological and biochemical approaches. The plasma membrane containing the putative plasma membrane aquaporin (PM-AQP) was prepared from tulip petals, phosphorylated in vitro, and used as the substrate for both of the purified PP2A preparations. Although both preparations dephosphorylated the phosphorylated PM-AQP at 20 °C, only the holo-type enzyme preparation acted at 5 °C on the phosphorylated PM-AQP with higher substrate specificity, suggesting that regulatory subunits are required for low temperature-dependent dephosphorylation of PM-AQP in tulip petals.

Heterologous Expression of Tulip Petal Plasma Membrane Aquaporins in Pichia pastoris for Water Channel Analysis

ABSTRACT Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs.

Functional characterization and hyperosmotic regulation of aquaporin in Synechocystis sp. PCC 6803

The genome of the cyanobacterium Synechocystis sp. PCC 6803 (hereafter, Synechocystis) contains an aqpZ gene (slr2057) which encodes an aquaporin (SsAqpZ), a membrane channel protein that might play a role in osmotic water transport and therefore the growth of Synechocystis. Structural characterization of SsAqpZ by protein sequence analysis and homology modelling revealed that it was more similar to bacterial aquaporin Z than the glycerol facilitator. To understand the functional role of SsAqpZ, the aqpZ knockout (KO) and myc-tagged aqpZ knockin (KI) Synechocystis were constructed. Water channel activity assays showed that SsAqpZ facilitated water transportation. SsAqpZ-mediated changes in cell volume were observed in wild-type (WT) and KI Synechocystis. Expression of SsAqpZ in KI Synechocystis was induced by extracellular hyperosmolarity. In the absence of hyperosmolarity, WT, KO and KI Synechocystis showed the same pattern of growth and no morphological or phenotypical perturbations. Under hyperosmotic condition, while the WT and also KI cells maintained a similar growth rate throughout the entire exponential phase, KO cells grew significantly slower. These results indicate that SsAqpZ has water channel activity and is involved in the adaptation and maintenance of growth of Synechocystis in a hyperosmotic environment.

Molecular cloning and sequence and 3D models analysis of the Sec61α subunit of protein translocation complex from Penicillium ochrochloron

The Sec61α subunit is the core subunit of the protein conducting channel which is required for protein translocation in eukaryotes and prokaryotes. In this study, we cloned a Sec61α subunit from Penicillium ochrochloron (PoSec61α). Sequence and 3D structural model analysis showed that PoSec61α conserved the typical characteristics of eukaryotic and prokaryotic Sec61α subunit homologues. The pore ring known as the constriction point of the channel is formed by seven hydrophobic amino acids. Two methionine residues from transmembrane α-helice 7 (TM7) contribute to the pore ring formation and projected notably to the pore area and narrowed the pore compared with the superposed residues at the corresponding positions in the crystal structures or the 3D models of the Sec61α subunit homologues in archaea or other eukaryotes, respectively. Results reported herein indicate that the pore ring residues differ among Sec61α subunit homologues and two hydrophobic residues in the TM7 contribute to the pore ring formation.