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

<p>Abstract</p> <p>Background</p> <p>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 <it>Atcngc5 </it>and <it>Atcngc10 </it>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

PRAF3 induces apoptosis and inhibits migration and invasion in human esophageal squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Prenylated Rab acceptor 1 domain family member 3 (PRAF3) is involved in the regulation of many cellular processes including apoptosis, migration and invasion. This study was conducted to investigate the effect of PRAF3 on apoptosis, migration and invasion in human esophageal squamous cell carcinoma (ESCC).</p> <p>Methods</p> <p>The expression of <it>PRAF3 </it>mRNA and protein in primary ESCC and the matched normal tissues (57cases) was determined by quantitative RT-PCR and Western blot. Immunohistochemical analysis of PRAF3 expression was carried out in paraffin-embedded sections of ESCC and correlated with clinical features. The role of PRAF3 in apoptosis, migration and invasion was studied in ESCC cell lines of Eca109 and TE-1 through the adenovirus mediated PRAF3 gene transfer. The effect of PRAF3 on apoptosis was analyzed by annexin V-FITC assay. The regulation of PRAF3 on migration was determined by transwell and wounding healing assay, while the cellular invasion was analyzed by matrigel-coated transwell assay.</p> <p>Results</p> <p>We found that the expression of PRAF3 was significantly down-regulated in ESCC tissue compared with the matched normal tissue and was correlated with the clinical features of pathological grade, tumor stage and lymph node metastasis. Moreover, overexpression of PRAF3 induced cell apoptosis through both caspase-8 and caspase-9 dependent pathways, and inhibited cell migration and invasion by suppressing the activity of both MMP-2 and MMP-9 in human ESCC cell lines.</p> <p>Conclusions</p> <p>Our data suggest that PRAF3 plays an important role in the regulation of tumor progression and metastasis and serves as a tumor suppressor in human ESCC. We propose that PRAF3 might be used as a potential therapeutic agent for human ESCC.</p

Subcellular distribution and expression of prenylated Rab acceptor 1 domain family, member 2 (PRAF2) in malignant glioma: Influence on cell survival and migration

Our previous studies revealed that the expression of the 19-kDa protein prenylated Rab acceptor 1 domain family, member 2 (PRAF2) is elevated in cancer tissues of the breast, colon, lung, and ovary, when compared to noncancerous tissues of paired samples. PRAF2 mRNA expression also correlated with several genetic and clinical features and is a candidate prognostic marker in the pediatric cancer neuroblastoma. The PRAF2-related proteins, PRAF1 and PRAF3, play multiple roles in cellular processes, including endo/exocytic vesicle trafficking and glutamate uptake. PRAF2 shares a high sequence homology with these family members, but its function remains unknown. In this study, we examined PRAF2 mRNA and protein expression in 20 different human cancer types using Affymetrix microarray and human tissue microarray (TMA) analyses, respectively. In addition, we investigated the subcellular distribution of PRAF2 by immunofluorescence microscopy and cell fractionation studies. PRAF2 mRNA and protein expression was elevated in several cancer tissues with highest levels in malignant glioma. At the molecular level, we detected native PRAF2 in small, vesicle-like structures throughout the cytoplasm as well as in and around cell nuclei of U-87 malignant glioma cells. We further found that monomeric and dimeric forms of PRAF2 are associated with different cell compartments, suggesting possible functional differences. Importantly, PRAF2 down-regulation by RNA interference significantly reduced the cell viability, migration, and invasiveness of U-87 cells. This study shows that PRAF2 expression is elevated in various tumors with exceptionally high expression in malignant gliomas, and PRAF2 therefore presents a candidate molecular target for therapeutic intervention. (Cancer Sci 2010