Arabidopsis protein disulfide isomerase-5 inhibits cysteine proteases during trafficking to vacuoles before programmed cell death of the endothelium in developing seeds.

Protein disulfide isomerase (PDI) oxidizes, reduces, and isomerizes disulfide bonds, modulates redox responses, and chaperones proteins. The Arabidopsis thaliana genome contains 12 PDI genes, but little is known about their subcellular locations and functions. We demonstrate that PDI5 is expressed in endothelial cells about to undergo programmed cell death (PCD) in developing seeds. PDI5 interacts with three different Cys proteases in yeast two-hybrid screens. One of these traffics together with PDI5 from the endoplasmic reticulum through the Golgi to vacuoles, and its recombinant form is functionally inhibited by recombinant PDI5 in vitro. Peak PDI5 expression in endothelial cells precedes PCD, whereas decreasing PDI5 levels coincide with the onset of PCD-related cellular changes, such as enlargement and subsequent collapse of protein storage vacuoles, lytic vacuole shrinkage and degradation, and nuclear condensation and fragmentation. Loss of PDI5 function leads to premature initiation of PCD during embryogenesis and to fewer, often nonviable, seeds. We propose that PDI5 is required for proper seed development and regulates the timing of PCD by chaperoning and inhibiting Cys proteases during their trafficking to vacuoles before PCD of the endothelial cells. During this transitional phase of endothelial cell development, the protein storage vacuoles become the de facto lytic vacuoles that mediate PCD.

The cyclic nucleotide gated cation channel AtCNGC10 traffics from the ER via Golgi vesicles to the plasma membrane of Arabidopsis root and leaf cells.

BACKGROUND: The cyclic nucleotide-gated ion channels (CNGCs) maintain cation homeostasis essential for a wide range of physiological processes in plant cells. However, the precise subcellular locations and trafficking of these membrane proteins are poorly understood. This is further complicated by a general deficiency of information about targeting pathways of membrane proteins in plants. To investigate CNGC trafficking and localization, we have measured Atcngc5 and Atcngc10 expression in roots and leaves, analyzed AtCNGC10-GFP fusions transiently expressed in protoplasts, and conducted immunofluorescence labeling of protoplasts and immunoelectron microscopic analysis of high pressure frozen leaves and roots. RESULTS: AtCNGC10 mRNA and protein levels were 2.5-fold higher in roots than leaves, while AtCNGC5 mRNA and protein levels were nearly equal in these tissues. The AtCNGC10-EGFP fusion was targeted to the plasma membrane in leaf protoplasts, and lightly labeled several intracellular structures. Immunofluorescence microscopy with affinity purified CNGC-specific antisera indicated that AtCNGC5 and AtCNGC10 are present in the plasma membrane of protoplasts. Immunoelectron microscopy demonstrated that AtCNGC10 was associated with the plasma membrane of mesophyll, palisade parenchyma and epidermal cells of leaves, and the meristem, columella and cap cells of roots. AtCNCG10 was also observed in the endoplasmic reticulum and Golgi cisternae and vesicles of 50-150 nm in size. Patch clamp assays of an AtCNGC10-GFP fusion expressed in HEK293 cells measured significant cation currents. CONCLUSION: AtCNGC5 and AtCNGC10 are plasma membrane proteins. We postulate that AtCNGC10 traffics from the endoplasmic reticulum via the Golgi apparatus and associated vesicles to the plasma membrane. The presence of the cation channel, AtCNGC10, in root cap meristem cells, cell plate, and gravity-sensing columella cells, combined with the previously reported antisense phenotypes of decreased gravitropic and cell enlargement responses, suggest roles of AtCNGC10 in modulating cation balance required for root gravitropism, cell division and growth.

The cyclic nucleotide-gated calmodulin-binding channel AtCNGC10 localizes to the plasma membrane and influences numerous growth responses and starch accumulation in Arabidopsis thaliana.

Cyclic nucleotide gated channels (CNGCs) that are regulated by calmodulin (CaM) have been shown to play essential roles in signal transduction, metabolism, and growth in animals. By contrast, very little is known about the subcellular location and the function of these channels in plants. Here we report on the effects of antisense suppression of the expression of AtCNGC10, a putative K+ channel, and the immunolocalization of the protein using an AtCNGC10-specific antiserum. In Arabidopsis thaliana leaves, AtCNGC10 was localized to the plasma membrane of mesophyll and parenchyma cells. Antisense AtCNGC10 plants had 40% of the AtCNGC10 mRNA levels and virtually undetectable protein levels relative to wild type plants. Antisense expression of AtCNGC10 did not affect the mRNA levels of AtCNGC13, the most closely related CNGC family member in the genome. Relative to wild type Columbia, antisense AtCNGC10 plants flowered 10 days earlier, and had a 25% reduction in leaf surface area, thickness and palisade parenchyma cell length. Their roots responded more slowly to gravitropic changes and the chloroplasts accumulated more starch. We propose that AtCNGC10, through interactions with CaM and cGMP, modulates cellular K+ balance across the plasma membrane, and that perturbations of this K+ gradient affect numerous growth and developmental processes.