Roles of COMM-domain-containing 1 in stability and recruitment of the copper-transporting ATPase in a mouse hepatoma cell line

A novel function of COMMD1 {COMM [copper metabolism MURR1 (mouse U2af1-rs1 region 1)]-domain-containing 1}, a protein relevant to canine copper toxicosis, was examined in the mouse hepatoma cell line Hepa 1-6 with multi-disciplinary techniques consisting of molecular and cellular biological techniques, speciation and elemental imaging. To clarify the function of COMMD1, COMMD1-knockdown was accomplished by introducing siRNA (small interfering RNA) into the cells. Although COMMD1-knockdown did not affect copper incorporation, it inhibited copper excretion, resulting in copper accumulation, which predominantly existed in the form bound to MT (metallothionein). It is known that the liver copper transporter Atp7b (ATP-dependent copper transporter 7beta), localizes on the trans-Golgi network membrane under basal copper conditions and translocates to cytoplasmic vesicles to excrete copper when its concentration exceeds a certain threshold, with the vesicles dispersing in the periphery of the cell. COMMD1-knockdown reduced the expression of Atp7b, and abolished the relocation of Atp7b back from the periphery to the trans-Golgi network membrane when the copper concentration was reduced by treatment with a Cu(I) chelator. The same phenomena were observed during COMMD1-knockdown when another Atp7b substrate, cis-diamminedichloroplatinum, and its sequestrator, glutathione ethylester, were applied. These results suggest that COMMD1 maintains the amount of Atp7b and facilitates recruitment of Atp7b from cytoplasmic vesicles to the trans-Golgi network membrane, i.e. COMMD1 is required to shuttle Atp7b when the intracellular copper level returns below the threshold

Cloning and functional characterization of a novel up-regulator, cartregulin, of carnitine transporter, OCTN2

Acetylcarnitine exerts therapeutic effects on some neurological disorders including Alzheimer’s disease. OCTN2 is known as a transporter for acetylcarnitine, but its expression in the brain is very low. To examine a brain-specific transporter for acetylcarnitine, we screened a rat brain cDNA library by hybridization using a DNA probe conserved among an OCTN family. A cDNA homologous to OCTN2 cDNA was isolated. The cDNA encoded a novel 146-amino acid protein with one putative transmembrane domain. The mRNA was expressed not only in rat brain but also in some other tissues. The novel protein was localized in endoplasmic reticulum when expressed in COS-7 cells but exhibited no transport activity for acetylcarnitine. However, when co-expressed with OCTN2, it enhanced the OCTN2-mediated transport by about twofold. The enhancement was accompanied by an increase in the levels of mRNA and protein. When OCTN2 was expressed in Xenopus oocytes by injection of its cRNA, its transport activity was enhanced by co-expression of the novel protein. These data suggest that the novel protein increases OCTN2 by stabilizing the mRNA in endoplasmic reticulum. The protein may be an up-regulator of OCTN2 and is tentatively designated cartregulin

Protein <i>N</i>-Myristoylation Plays a Critical Role in the Endoplasmic Reticulum Morphological Change Induced by Overexpression of Protein Lunapark, an Integral Membrane Protein of the Endoplasmic Reticulum

<div><p><i>N</i>-myristoylation of eukaryotic cellular proteins has been recognized as a modification that occurs mainly on cytoplasmic proteins. In this study, we examined the membrane localization, membrane integration, and intracellular localization of four recently identified human <i>N</i>-myristoylated proteins with predicted transmembrane domains. As a result, it was found that protein Lunapark, the human ortholog of yeast protein Lnp1p that has recently been found to be involved in network formation of the endoplasmic reticulum (ER), is an <i>N</i>-myristoylated polytopic integral membrane protein. Analysis of tumor necrosis factor-fusion proteins with each of the two putative transmembrane domains and their flanking regions of protein Lunapark revealed that transmembrane domain 1 and 2 functioned as type II signal anchor sequence and stop transfer sequence, respectively, and together generated a double-spanning integral membrane protein with an N-/C-terminal cytoplasmic orientation. Immunofluorescence staining of HEK293T cells transfected with a cDNA encoding protein Lunapark tagged with FLAG-tag at its C-terminus revealed that overexpressed protein Lunapark localized mainly to the peripheral ER and induced the formation of large polygonal tubular structures. Morphological changes in the ER induced by overexpressed protein Lunapark were significantly inhibited by the inhibition of protein <i>N</i>-myristoylation by means of replacing Gly2 with Ala. These results indicated that protein <i>N</i>-myristoylation plays a critical role in the ER morphological change induced by overexpression of protein Lunapark.</p></div

Structure of protein Lunapark.

<p>Alignment of the N-terminal sequences and the transmembrane domains (TMDs) of the Lunapark family of proteins is shown. <i>N</i>-myristoylation motifs are shown in red in the N-terminal sequence. Hydrophobic amino acids are shown in grey and charged amino acids in blue in the transmembrane domain and their flanking regions. The predicted transmembrane domains are indicated as solid lines.</p

Detection of protein <i>N</i>-myristoylation in <i>in vitro</i>-synthesized protein Lunapark by MS analysis.

<p>A. Purified <i>in vitro</i>-synthesized protein Lunapark (1 µg) was separated by electrophoresis in an SDS-PAGE gel (12.5%). B. MALDI-MS of the tryptic peptides from <i>in vitro-</i>synthesized protein Lunapark. The peaks of the tryptic peptides derived from <i>in vitro</i>-synthesized protein Lunapark are indicated by stars. C. MS/MS analysis was performed for the peak at m/z 846.55. The identified N-myristoylated N-terminal sequence is shown.</p

Role of the zinc finger motif of protein Lunapark in the ER morphological change induced by protein Lunapark.

<p>A. Alignment of the zinc finger motif of Lunapark protein family members. Highly conserved cysteine residues are indicated by red arrows. B. Detection of protein <i>N</i>-myristoylation of Lunapark-CtoA-FLAG expressed in HEK293T cells. cDNAs encoding Lunapark-FLAG, Lunapark-G2A-FLAG, and Lunapark-CtoA-FLAG were transfected in to HEK293T cells, and their expression and the <i>N</i>-myristoylation of the products in the total cell lysates were evaluated by Western blotting analysis and [³H]myristic acid ([³H]Myr) labeling, respectively. C. Intracellular localization of Lunapark-FLAG, Lunapark-G2A-FLAG, and Lunapark-CtoA-FLAG was determined by immunofluorescence staining of HEK293T cells transfected with cDNAs encoding these three proteins using an anti-FLAG antibody. Right panel shows a close-up view of the area surrounded by a white box in the immunofluorescence image. D. Quantitative analysis of the ER morphological change in HEK293T cells induced by Lunapark-FLAG (WT), Lunapark-G2A-FLAG (G2A), and Lunapark-CtoA-FLAG (CtoA). cDNAs encoding Lunapark-FLAG, Lunapark-G2A-FLAG, and Lunapark-CtoA-FLAG were transfected in to HEK293T cells and the morphological change of the ER in each cell was determined by immunofluorescence staining and the extent of the ER morphological change was compared. The extent of ER morphological changes is expressed as a percentage of the number of cells with highly tubular, partially tubular, and non-tubular ER against the total number of transfected cells. Data are expressed as mean ± SD for five independent experiments. **<i>P</i><0.001 vs. WT. *<i>P</i><0.01 vs. WT.</p

Analysis of membrane integration and membrane topology of protein Lunapark.

<p>A. Structure of pro-GLC-TNF, Lunapark-TM1-GLC-TNF, and Lunapark-TM1-G2A-GLC-TNF for analysis of the function of transmembrane domain 1 (TM1) of protein Lunapark. B. cDNAs encoding pro-GLC-TNF, Lunapark-TM1-GLC-TNF were transfected in to COS-1 cells, and their secretion and expression in total cell lysates were evaluated by Western blotting analysis using an anti-TNF antibody. <i>N</i>-glycosylation of the expressed proteins in total cell lysates was determined by the change in its molecular weight after treatment with glycopeptidase F (GPF). S, cell culture supernatant; T, total cell lysates. C. cDNAs encoding Lunapark-TM1-GLC-TNF and Lunapark-TM1-G2A-GLC-TNF were transfected in to COS-1 cells, and their expression and the <i>N</i>-myristoylation of the products in total cell lysates were evaluated by Western blotting analysis and [³H]myristic acid ([³H]Myr) labeling, respectively. <i>N</i>-glycosylation of the expressed protein in total cell lysates was determined by the change in molecular weight after treatment with GPF. D. Structure of Lunapark-TM1-GLC-TNF, Lunapark-TM1/2-GLC-TNF, and Lunapark-GLC-TNF for analysis of the function of transmembrane domain 2 (TM2) of protein Lunapark. E. cDNAs encoding Lunapark-TM1-GLC-TNF, Lunapark-TM1/2-GLC-TNF, and Lunapark-GLC-TNF were transfected in to COS-1 cells, and their expression in total cell lysates was evaluated by Western blotting analysis using an anti-TNF antibody. <i>N</i>-glycosylation of the expressed proteins in total cell lysates was determined by the change in molecular weight after treatment with GPF. F. Schematic representation of the transmembrane topology of protein Lunapark in the ER membrane.</p