A novel choline cotransporter sequestration compartment in cholinergic neurons revealed by selective endosomal ablation.

The sodium-dependent, high affinity choline transporter - choline cotransporter - (ChCoT, aka: cho-1, CHT1, CHT) undergoes constitutive and regulated trafficking between the plasma membrane and cytoplasmic compartments. The pathways and regulatory mechanisms of this trafficking are not well understood. We report herein studies involving selective endosomal ablation to further our understanding of the trafficking of the ChCoT. Selective ablation of early sorting and recycling endosomes resulted in a decrease of approximately 75% of [3H]choline uptake and approximately 70% of [3H]hemicholinium-3 binding. Western blot analysis showed that ablation produced a similar decrease in ChCoTs in the plasma membrane subcellular fraction. The time frame for this loss was approximately 2 h which has been shown to be the constitutive cycling time for ChCoTs in this tissue. Ablation appears to be dependent on the intracellular cycling of transferrin-conjugated horseradish peroxidase and the selective deposition of transferrin-conjugated horseradish peroxidase in early endosomes, both sorting and recycling. Ablated brain slices retained their capacity to recruit via regulated trafficking ChCoTs to the plasma membrane. This recruitment of ChCoTs suggests that the recruitable compartment is distinct from the early endosomes. It will be necessary to do further studies to identify the novel sequestration compartment supportive of the ChCoT regulated trafficking.

Regulation of ANKRD9 expression by lipid metabolic perturbations.

Fatty acid oxidation (FAO) defects cause abnormal lipid accumulation in various tissues, which provides an opportunity to uncover novel genes that are involved in lipid metabolism. During a gene expression study in the riboflavin deficient induced FAO disorder in the chicken, we discovered the dramatic increase in mRNA levels of an uncharacterized gene, ANKRD9. No functions have been ascribed to ANKRD9 and its orthologs, although their sequences are well conserved among vertebrates. To provide insight into the function of ANKRD9, the expression of ANKRD9 mRNA in lipidperturbed paradigms was examined. The hepatic mRNA level of ANKRD9 was repressed by thyroid hormone (T(3)) and fasting, elevated by re-feeding upon fasting. However, ANKRD9 mRNA level is reduced in response to apoptosis. Transient transfection assay with green fluorescent protein tagged- ANKRD9 showed that this protein is localized within the cytoplasm. These findings point to the possibility that ANKRD9 is involved in intracellular lipid accumulation. [BMB reports 2009; 42(9): 568-573].

Cloning and partial characterization of four plasmalemmal-associated syntaxin isoforms in Limulus.

We describe herein the cloning of a group of syntaxins in Limulus that are associated with the plasma membrane. Initially, multiple degenerate oligonucleotide primers (DOP) and probes were designed from sequences of known plasma membrane associated syntaxins. Combined experiments using reverse transcriptase-polymerase chain reaction (RT-PCR), colony hybridization and reverse dot blot yielded three distinct probes. Subsequently, two cDNA libraries derived from the Limulus central nervous system (CNS) were screened and four distinct isoforms, designated Limulus syntaxin (Lim-syn) 1A, 1B, 1C and 1D, were obtained from forty cloned full-length sequences. The predicted amino acid (aa) sequences 1-265 were identical for Lim-syn 1A, 1C and for Lim-syn 1B, 1D, respectively. A comparison of the 265 aa cytoplasmic segments for the two subgroups Lim-syn 1A/1C and Lim-syn 1B/1D differed at 13 aa residues within this sequence. Lim-syn 1A and 1B contained 290 aa residues, and both contained a transmembrane domain (TMD, 267-288) and a myristylation-like site (286-290) at the C-termini. Lim-syn 1C (291 residues) contained only the TMD whereas Lim-syn 1D was truncated (277 residues) and had neither a TMD nor a myristylation-like site. All Lim-syn isoforms showed great identity with syntaxin 1-homologs (syntaxin 1A/1B) from various other species. Ribonuclease protection assay (RPA) analyses revealed distinctive expression patterns for individual Lim-syn transcripts but all were detectable in the CNS. Moreover, the antibody (anti-Lim-syn-1) produced against aa 133-145 epitope of Lim-syn identified a protein of approximately 35 kDa found only in CNS tissues.

Molecular cloning and identification of a putative PKC epsilon cDNA from Limulus polyphemus brain.

The protein kinase C (PKC) family of enzymes is broadly distributed and has been implicated in a diverse array of cellular functions. Recent evidence supporting PKC involvement in the regulation of the Limulus choline cotransporter prompted us to clone PKC from a Limulus central nervous system (CNS) cDNA library. An Aplysia californica calcium independent PKC (Apl II) cDNA probe was used to screen the library and 5' RACE SMART PCR was used to obtain the full-length sequence. The resulting cDNA, which included 5' and 3' nontranslation regions, was 4675 bp. Analysis of the encoded peptide sequence using the Swiss-prot database revealed at least 58% identity to PKC epsilon. A commercial polyclonal antibody against PKC epsilon was used in Western blots to positively label a 30 kDa protein from Limulus CNS and the expressed fusion protein of the encoded sequence. These data support the presence of a newly identified PKC-like homolog in Limulus which likely represents a PKC epsilon equivalent.