Arowu R. Tanaka

ATP Binding/Hydrolysis by and Phosphorylation of Peroxisomal ATP-binding Cassette Proteins PMP70 (ABCD3) and Adrenoleukodystrophy Protein (ABCD1)

The 70-kDa peroxisomal membrane protein (PMP70) and adrenoleukodystrophy protein (ALDP), half-size ATP-binding cassette transporters, are involved in metabolic transport of long and very long chain fatty acids into peroxisomes. We examined the interaction of peroxisomal ATP-binding cassette transporters with ATP using rat liver peroxisomes. PMP70 was photoaffinity-labeled at similar efficiencies with 8-azido-[α-32P]ATP and 8-azido-[γ-32P]ATP when peroxisomes were incubated with these nucleotides at 37 °C in the absence Mg2+ and exposed to UV light without removing unbound nucleotides. The photoaffinity-labeled PMP70 and ALDP were co-immunoprecipitated together with other peroxisomal proteins, which also showed tight ATP binding properties. Addition of Mg2+ reduced the photoaffinity labeling of PMP70 with 8-azido-[γ-32P]ATP by 70%, whereas it reduced photoaffinity labeling with 8-azido-[α-32P]ATP by only 20%. However, two-thirds of nucleotide (probably ADP) was dissociated during removal of unbound nucleotides. These results suggest that ATP binds to PMP70 tightly in the absence of Mg2+, the bound ATP is hydrolyzed to ADP in the presence of Mg2+, and the produced ADP is dissociated from PMP70, which allows ATP hydrolysis turnover. Properties of photoaffinity labeling of ALDP were essentially similar to those of PMP70. Vanadate-induced nucleotide trapping in PMP70 and ALDP was not observed. PMP70 and ALDP were also phosphorylated at a tyrosine residue(s). ATP binding/hydrolysis by and phosphorylation of PMP70 and ALDP are involved in the regulation of fatty acid transport into peroxisomes.

Verapamil Increases the Apolipoprotein-Mediated Release of Cellular Cholesterol by Induction of ABCA1 Expression Via Liver X Receptor-Independent Mechanism

Objective—Release of cellular cholesterol and phospholipid mediated by helical apolipoprotein and ATP-binding cassette transporter (ABC) A1 is a major source of plasma HDL. We investigated the effect of calcium channel blockers on this reaction. Methods and Results—Expression of ABCA1, apoA-I–mediated cellular lipid release, and HDL production were enhanced in cAMP analogue-treated RAW264 cells by verapamil, and similar effects were also observed with other calcium channel blockers. The verapamil treatment resulted in rapid increase in ABCA1 protein and its mRNA, but not the ABCG1 mRNA, another target gene product of the nuclear receptor liver X receptor (LXR). By using the cells transfected with a mouse ABCA1 promoter–luciferase construct (−1238 to +57bp), verapamil was shown to enhance the transcriptional activity. However, it did not increase transcription of LXR response element-driven luciferase vector. Conclusions—The data demonstrated that verapamil increases ABCA1 expression through LXR-independent mechanism and thereby increases apoA-I–mediated cellular lipid release and production of HDL.

Cloning, characterization and tissue distribution of the rat ATP-binding cassette (ABC) transporter ABC2/ABCA2.

The ABC1 (ABCA) subfamily of the ATP-binding cassette (ABC) transporter superfamily has a structural feature that distinguishes it from other ABC transporters. Here we report the cloning, molecular characterization and tissue distribution of ABC2/ABCA2, which belongs to the ABC1 subfamily. Rat ABC2 is a protein of 2434 amino acids that has 44.5%, 40.0% and 40.8% identity with mouse ABC1/ABCA1, human ABC3/ABCA3 and human ABCR/ABCA4 respectively. Immunoblot analysis showed that proteins of 260 and 250 kDa were detected in COS-1 cells transfected with ABC2 having a haemagglutinin tag, while no band was detected in mock-transfected cells. After incubation with N-glycosidase F, the mobilities of the two proteins increased and a single band was detected, suggesting that ABC2 is a glycoprotein. Photoaffinity labelling with 8-azido-[alpha-(32)P]ATP confirmed that ATP binds to the ABC2 protein in the presence of Mg(2+). RNA blot analysis showed that ABC2 mRNA is most abundant in rat brain. Examination of brain by in situ hybridization determined that ABC2 is expressed at high levels in the white matter, indicating that it is expressed in the oligodendrocytes. ABC2, therefore, is a glycosylated ABC transporter protein, and may play an especially important role in the brain. In addition, the N-terminal 60-amino-acid sequence of the human ABC1, which was missing from previous reports, has been determined.

The maintenance of the endoplasmic reticulum network is regulated by p47, a cofactor of p97, through phosphorylation by cdc2 kinase

The endoplasmic reticulum (ER) has a characteristic complex polygonal structure with hallmark three‐way junctions in many types of cells. To investigate the mechanisms responsible for maintaining the ER network, we established ER disassembly and reassembly assays in semi‐intact Chinese hamster ovary (CHO) cells that constitutively expressed heat shock protein‐47 fused to the green fluorescent protein (GFP‐HSP47) as an ER marker (the cells are referred to as CHO‐HSP cells). Using these assays, we found that maintenance of the ER network required cytosol and adenosine triphosphate/guanosine 5′‐triphosphate (ATP/GTP) hydrolysis, but not actin filaments or microtubules. We also showed that the ER network was disrupted upon addition of either N‐ethylmaleimide‐treated cytosol after washing semi‐intact cells with high salt solution or mitotic cytosol in nocodazole‐treated semi‐intact CHO‐HSP cells. The disrupted ER network induced by mitotic cytosol was reformed by the addition of interphase cytosol. In addition, we found that p47, a cofactor of p97, was essential for the maintenance of the ER network, and that phosphorylation of p47 by cdc2 kinase resulted in ER network disruption by mitotic cytosol. Taken together, these results imply that the maintenance of the ER network requires a membrane fusion process mediated by p97/p47, and that cell cycle‐dependent morphological changes of the ER network are regulated through phosphorylation/dephosphorylation of p47.

The ABCA1 Q597R mutant undergoes trafficking from the ER upon ER stress

Mutations in ATP binding cassette transporter 1 (ABCA1), a membrane protein associated with cellular cholesterol efflux, cause Tangier disease (TD). Previously, we showed that an ABCA1 Q597R mutant (QR) identified in TD is retained in the endoplasmic reticulum. Here, we report that QR trafficking to the plasma membrane was rapidly induced by thapsigargin or DTT, indicating that ER stress-induced QR trafficking. However, pharmacological rescue of ABCA1 activity was not observed. The trafficking was dependent on COPII components and occurred via the ER-Golgi intermediate compartments. Furthermore, we found that QR was more sensitive to ER stress than ATF6, a transcription factor associated with the ER stress response. These results suggest that thapsigargin can be effective in correcting trafficking defects, and raise the possibility that ER stress-induced trafficking is involved in ER quality control.

Formation of cholesterol-enriched structures by aberrant intracellular accumulation of ATP-binding cassette transporter A1

ATP‐binding cassette transporter A1 (ABCA1) is a key transporter associated with excess cellular lipid efflux. Here, we report that in HEK293 cells ABCA1 functions in intracellular compartments along the endocytic pathway. Inhibition of ABCA1‐GFP degradation with proteasome inhibitors induced the internalization of ABCA1 and the formation of intracellular round‐shaped structures, designated “A1 bodies”. Importantly, cholesterol was selectively accumulated in A1 bodies, and this depended on the cholesterol efflux activity of ABCA1. Treatment with either lactacystin or acetylated LDL, which reduces proteasome activity, resulted in internalization of ABCA1 in mouse peritoneal macrophages. By performing array analysis on macrophages treated with these reagents, we identified Rab4 as a key protein involved in the internalization and aberrant accumulation of ABCA1 in HEK cells. Treatment of the cells with proteasome inhibitors inhibited the degradation of Rab4, and Rab4 over‐expression induced the formation of small A1 bodies. Furthermore, A1 bodies formation was substantially inhibited by silencing of the endogenous Rab4 gene. Taken together, our findings suggest that the endocytic ABCA1 possesses cholesterol efflux activity, and thus the cellular control of post‐endocytic sorting, retention or recycling of functional ABCA1 in the endocytic vesicles, which is in part regulated by Rab4, is important for cholesterol metabolism in living cells.