Intramolecular rearrangements as a consequence of the dephosphorylation of phosphoaspartate residues in proteins

Sedlin is an evolutionarily conserved protein encoded by the causative gene SEDL for spondyloepiphyseal dysplasia tarda. Nevertheless, how Sedlin mutations cause the disease remains unknown. Here, the intracellular chloride channel protein CLIC1 was shown to associate with Sedlin by yeast two-hybrid screening. Green fluorescence protein-CLIC1 readily co-immunoprecipitated with FLAG-Sedlin. In addition, both proteins colocalized extensively in cytoplasmic vesicular/reticular structures in COS-7 cells, suggesting their interaction at intracellular membranous organelles. Sedlin also associated with CLIC2 in yeast two-hybrid assays. The link between Sedlin and the intracellular chloride channels is the first step to understand their functional interplays.     

PAM14, a novel MRG- and Rb-associated protein, is not required for development and T-cell function in mice

PAM14 has been found to associate in complexes with the MORF4/MRG family of proteins as well as Rb, the tumor suppressor protein. This suggested that it might be involved in cell growth, immortalization, and/or senescence. To elucidate the in vivo function of PAM14, we characterized the expression pattern of mouse Pam14 and generated PAM14-deficient (Pam14(-/-)) mice. Pam14 was widely expressed in all mouse tissues and as early as 7 days during embryonic development. Despite this ubiquitous expression in wild-type mice, Pam14(-/-) mice were healthy and fertile. Response to mitogenic stimulation and production of interleukin-2 were the same in stimulated splenic T cells from Pam14(-/-) mice as in control littermates. Cell growth rates of mouse embryonic fibroblasts (MEFs) from all three genotypes were the same, and immortalized cells were obtained from all cell cultures during continuous culture. There was also no difference in expression of growth-related genes in response to serum stimulation in the null versus control MEFs. These data demonstrate that PAM14 is not essential for normal mouse development and cell cycle control. PAM14 likely acts as an adaptor protein in nucleoprotein complexes and is probably compensated for by another functionally redundant protein(s).     

The organellular chloride channel protein CLIC4/mtCLIC translocates to the nucleus in response to cellular stress and accelerates apoptosis

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to the mitochondria and cytoplasm of keratinocytes and participates in the apoptotic response to stress. We now show that multiple stress inducers cause the translocation of cytoplasmic CLIC4 to the nucleus. Immunogold electron microscopy and confocal analyses indicate that nuclear CLIC4 is detected prior to the apoptotic phenotype. CLIC4 associates with the Ran, NTF2, and Importin-alpha nuclear import complexes in immunoprecipitates of lysates from cells treated with apoptotic/stress-inducing agents. Deletion or mutation of the nuclear localization signal in the C terminus of CLIC4 eliminates nuclear translocation, whereas N terminus deletion enhances nuclear localization. Targeting CLIC4 to the nucleus via adenoviral transduction accelerates apoptosis when compared with cytoplasmic CLIC4, and only nuclear-targeted CLIC4 causes apoptosis in Apaf null mouse fibroblasts or in Bcl-2-overexpressing keratinocytes. These results indicate that CLIC4 nuclear translocation is an integral part of the cellular response to stress and may contribute to the initiation of nuclear alterations that are associated with apoptosis.     

Identification of a novel member of the chloride intracellular channel gene family (CLIC5) that associates with the actin cytoskeleton of placental microvilli

The chloride intracellular channel (CLIC) gene family has been implicated in chloride ion transport within various subcellular compartments. We report here the molecular, biochemical, and cellular characterization of a new member of this gene family termed CLIC5. CLIC5 was isolated from extracts of placental microvilli as a component of a multimeric complex consisting of several known cytoskeletal proteins, including actin, ezrin, alpha-actinin, gelsolin, and IQGAP1. We cloned human cDNAs and generated antibodies specific for CLIC5, CLIC1/NCC27, and CLIC4/huH1/p64H1. CLIC5 shares 52-76% overall identity with human CLIC1, CLIC2, CLIC3, and CLIC4. Northern blot analysis showed that CLIC5 has a distinct pattern of expression compared with CLIC1 and CLIC4. Immunoblot analysis of extracts from placental tissues demonstrated that CLIC4 and CLIC5 are enriched in isolated placental microvilli, whereas CLIC1 is not. Moreover, in contrast to CLIC1 and CLIC4, CLIC5 is associated with the detergent-insoluble cytoskeletal fraction of microvilli. Indirect immunofluorescence microscopy revealed that CLIC4 and CLIC5 are concentrated within the apical region of the trophoblast, whereas CLIC1 is distributed throughout the cytoplasm. These studies suggest that CLIC1, CLIC4, and CLIC5 play distinct roles in chloride transport and that CLIC5 interacts with the cortical actin cytoskeleton in polarized epithelial cells.     

Trs20 is Required for TRAPP II Assembly

The modular TRAPP complexes act as nucleotide exchangers to activate the Golgi Ypt/Rab GTPases, Ypt1 and Ypt31/Ypt32. In yeast, TRAPP I acts at the cis‐Golgi and its assembly and structure are well characterized. In contrast, TRAPP II acts at the trans‐Golgi and is poorly understood. Especially puzzling is the role of Trs20, an essential TRAPP I/II subunit required neither for the assembly of TRAPP I nor for its Ypt1‐exchange activity. Mutations in Sedlin, the human functional ortholog of Trs20, cause the cartilage‐specific disorder SEDT. Here we show that Trs20 interacts with the TRAPP II‐specific subunit Trs120. Furthermore, the Trs20‐Trs120 interaction is required for assembly of TRAPP II and for its Ypt32‐exchange activity. Finally, Trs20‐D46Y, with a single‐residue substitution equivalent to a SEDT‐causing mutation in Sedlin, interacts with TRAPP I, but the resulting TRAPP complex cannot interact with Trs120 and TRAPP II cannot be assembled. These results indicate that Trs20 is crucial for assembly of TRAPP II, and the defective assembly caused by a SEDT‐linked mutation suggests that this role is conserved .     

Generation and characterization of mice with null mutation of the chloride intracellular channel 1 gene

CLIC1 belongs to a family of highly conserved and widely expressed intracellular chloride ion channel proteins existing in both soluble and membrane integrated forms. To study the physiological and biological role of CLIC1 in vivo, we undertook conditional gene targeting to engineer Clic1 gene knock‐out mice. This represents creation of the first gene knock‐out of a vertebrate CLIC protein family member. We first generated a Clic1 Knock‐in (Clic1^FN) allele, followed by Clic1 knock‐out (Clic1^−/−) mice by crossing Clic1^FN allele with TNAP‐cre mice, resulting in germline gene deletion through Cre‐mediated recombination. Mice heterozygous or homozygous for these alleles are viable and fertile and appear normal. However, Clic1^−/− mice show a mild platelet dysfunction characterized by prolonged bleeding times and decreased platelet activation in response to adenosine diphosphate stimulation linked to P2Y₁₂ receptor signaling. genesis 48:127–136, 2010. © 2010 Wiley‐Liss, Inc.