ABCA1 mediates release of cellular cholesterol and phospholipid to form high density lipoprotein (HDL).The three different mutants in the first extracellular domain of human ABCA1 associated with Tangier disease, R587W, W590S, and Q597R, were examined for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. ABCA1-GFP expressed in HEK293 was fully functional for apoA-I-mediated HDL assembly. Immunostaining and confocal microscopic analyses demonstrated that ABCA1-GFP was mainly localized to the plasma membrane (PM) but also substantially in intracellular compartments. All three mutant ABCA1-GFPs showed no or little apoA-I-mediated HDL assembly. R587W and Q597R were associated with impaired processing of oligosaccharide from high mannose type to complex type and failed to be localized to the PM, whereas W590S did not show such dysfunctions. Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant. Photoaffinity labeling of W590S with 8-azido-[␣-32 P]ATP was stimulated by adding ortho-vanadate in the presence of Mn 2؉ as much as in the presence of wildtype ABCA1. These results suggest that the defect of HDL assembly in R587W and Q597R is due to the impaired localization to the PM, whereas W590S has a functional defect other than the initial ATP binding and hydrolysis.Cholesterol is not catabolized in the peripheral cells and therefore mostly released and transported to the liver for conversion to bile acids to maintain cholesterol homeostasis. The same pathway may also remove cholesterol that has pathologically accumulated in the cells such as an initial stage of atherosclerosis. Assembly of high density lipoprotein (HDL) 1 particles by helical apolipoproteins with cellular lipid has been recognized as one of the major mechanisms for cellular cholesterol release (1, 2). The importance of this active cholesterolreleasing pathway in regulating cholesterol homeostasis became apparent by the finding that it is impaired in the cells from patients with Tangier disease, a genetic deficiency of circulating HDL (3, 4). Mutations were identified in ATP-binding cassette transporter A1 (ABCA1) of the Tangier disease (TD) patients (5-7), but the molecular mechanism of ABCA1 in the apolipoprotein-mediated HDL assembly remains unclear. Although direct interaction between ABCA1 and apoA-I at the cell surface has been suggested on the basis of chemical crosslinking experiments (8, 9), an indirect role of ABCA1 in the apoA-I binding to the cell was also proposed by a model that ABCA1 induces phosphatidylserine exofacial flopping to generate the microenvironment required for the docking of apoA-I at the cell surface (10). The predominant substrates of the ABCA1-mediated lipid release reaction are still to be determined for the HDL assembly reaction (11, 12). More than 30 mutations have been mapped in the ABCA1 gene in patients with familial hypoalphalipoproteinemia (FHA) and TD (5-7, 13-15). Many mutations have been ...

Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various physical extremes. Although trehalose and late embryogenesis abundant (LEA) proteins have been extensively studied as potent protectants against dehydration in other anhydrobiotic organisms, tardigrades produce high amounts of tardigrade-unique protective proteins. Cytoplasmic-abundant heat-soluble (CAHS) proteins are uniquely invented in the lineage of eutardigrades, a major class of the phylum Tardigrada and are essential for their anhydrobiotic survival. However, the precise mechanisms of their action in this protective role are not fully understood. In the present study, we first postulated the presence of tolerance proteins that form protective condensates via phase separation in a stress-dependent manner and searched for tardigrade proteins that reversibly form condensates upon dehydration-like stress. Through a comprehensive search using a desolvating agent, trifluoroethanol (TFE), we identified 336 proteins, collectively dubbed “TFE-Dependent ReversiblY condensing Proteins (T-DRYPs).” Unexpectedly, we rediscovered CAHS proteins as highly enriched in T-DRYPs, 3 of which were major components of T-DRYPs. We revealed that these CAHS proteins reversibly polymerize into many cytoskeleton-like filaments depending on hyperosmotic stress in cultured cells and undergo reversible gel-transition in vitro. Furthermore, CAHS proteins increased cell stiffness in a hyperosmotic stress-dependent manner and counteract the cell shrinkage caused by osmotic pressure, and even improved the survival against hyperosmotic stress. The conserved putative helical C-terminal region is necessary and sufficient for filament formation by CAHS proteins, and mutations disrupting the secondary structure of this region impaired both the filament formation and the gel transition. On the basis of these results, we propose that CAHS proteins are novel cytoskeleton-like proteins that form filamentous networks and undergo gel-transition in a stress-dependent manner to provide on-demand physical stabilization of cell integrity against deformative forces during dehydration and could contribute to the exceptional physical stability in a dehydrated state.

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We observed the disassembly of endoplasmic reticulum (ER) exit sites (ERES) by confocal microscopy during mitosis in Chinese hamster ovary (CHO) cells by using Yip1A fused to green fluorescence protein (GFP) as a transmembrane marker of ERES. Photobleaching experiments revealed that Yip1A-GFP, which was restricted to the ERES during interphase, diffused throughout the ER network during mitosis. Next, we reconstituted mitotic disassembly of Yip1A-GFP-labeled ERES in streptolysin O-permeabilized CHO cells by using mitotic L5178Y cytosol. Using the ERES disassembly assay and the anterograde transport assay of GFP-tagged VSVGts045, we demonstrated that the phosphorylation of p47 by Cdc2 kinase regulates the disassembly of ERES and results in the specific inhibition of ER-to-Golgi transport during mitosis. INTRODUCTIONThe first step of anterograde transport from the endoplasmic reticulum (ER) to the Golgi apparatus is the recruitment of the cytosolic coat complex (COP II) to specialized sites of ER export, called ER exit sites (ERES) or transitional ER (tER), where cargo proteins are actively sorted and concentrated in COP II-coated vesicles (Hobman et al., 1998;Hong, 1998;Kaiser and Ferro-Novick, 1998;Stephens et al., 2000;Muniz et al., 2001;Aridor et al., 2001;Barlowe, 2002). Morphologically, this compartment was originally defined as a ribosome-free ER subdomain that is continuous with the rough ER and contains protrusions resembling budding vesicles (Merisko et al., 1986;Orci et al., 1991;Rossanese et al., 1999;Shugrue et al., 1999). Because ERES are starting points for ER-to-Golgi transport, the balance of which influences Golgi morphology, the biogenesis of ERES is thought to be closely coupled to Golgi biogenesis (Ward et al., 2001;Bevis et al., 2002). Thus, ERES are maintained as an important subdomain of the ER, not only for the sorting/budding of proteins from the ER but also for Golgi biogenesis. However, less is known about the mechanism by which ERES maintain their distinct morphological identity as subdomains within the general ER or how their formation/disassembly is regulated during the cell cycle. Few studies have examined either ERES dynamics within the cell or the biochemical requirements for ERES formation.Time-lapse imaging has been used to study the dynamic behavior of ERES by using fluorescence-tagged cytosolic COP II components as probes, such as in Pichia pastoris or Chinese hamster ovary (CHO) cells by using Sec13-GFP (Hammond and Glick, 2000;Bevis et al., 2002), or in HeLa cells by using Sec23A-YFP (Stephens, 2003). These studies revealed that ERES are long-lived compartments that move slowly on the ER network and with apparently restricted mobility. Despite the limited movement of individual ERES, fusion, fission, or de novo formation of ERES can be seen in interphase HeLa cells (Stephens, 2003). The cell cycle-dependent accumulation of ERES also has been studied in mammalian cells and yeast. Immunofluorescence studies using anti-Sec13 antibodies revealed that ERES grew in number during i...

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 photoaffinitylabeled at similar efficiencies with 8-azido-[␣-32 P]ATP and 8-azido-[␥-32 P]ATP when peroxisomes were incubated with these nucleotides at 37°C in the absence Mg 2؉ 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 Mg 2؉ reduced the photoaffinity labeling of PMP70 with 8-azido-[␥-32 P]ATP by 70%, whereas it reduced photoaffinity labeling with 8-azido-[␣-32 P]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 Mg 2؉ , the bound ATP is hydrolyzed to ADP in the presence of Mg 2؉ , 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.ATP-binding cassette (ABC) 1 superfamily proteins are composed of two homologous halves, each of which typically contains six transmembrane ␣ helices and a nucleotide binding fold (NBF). Prominent members of eukaryotic ABC superfamily proteins, such as the multidrug efflux pump MDR1 (ABCB1) and the cystic fibrosis transmembrane conductance regulator CFTR (ABCC7), are full size and contain 12 transmembrane ␣ helices and 2 NBFs. On the other hand, most of the organelle ABC superfamily proteins, such as antigen transporters TAP1 (ABCB1) and TAP2 (ABCB2) on endoplasmic reticulum membranes and peroxisomal ABC proteins, are half size and contain six transmembrane ␣ helices and one NBF.To date, four peroxisomal ABC proteins have been identified in mammalian peroxisomes: the 70-kDa peroxisomal membrane protein (PMP70, ABCD3), adrenoleukodystrophy protein (ALDP, ABCD1), ALDP-related protein (ALDRP, ABCD2), and PMP70-related protein (P70R, ABCD4) (1-7). These half-size ABC proteins are supposed to work after dimerization. Disruption of the half-size ABC protein gene of Saccharomyces cerevisiae PAT1 (Pxa1) or PAT2 (Pxa2), whose products were identified on peroxisomes, resulted in impaired growth in oleic acid medium, suggesting that Pat1p and Pat2p function as heterodimers (8 -11). Indeed, Liu et al. (12) have shown that homoas well as heterodimerization occurred among the ALDP, AL-DRP, and PMP70 by using the yeast two-hybrid syste...

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 Nethylmaleimide-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 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.