Mucolipin-1 (MLN1) is a membrane protein with homology to the transient receptor potential channels and other non-selective cation channels. It is encoded by the MCOLN1 gene, which is mutated in patients with mucolipidosis type IV (MLIV), an autosomal recessive disease that is characterized by severe abnormalities in neurological development as well as by ophthalmologic defects. At the cellular level, MLIV is associated with abnormal lysosomal sorting and tra⁄cking. Here we identify the channel function of human MLN1 and characterize its properties. MLN1 represents a novel Ca 2+ -permeable channel that is transiently modulated by changes in [Ca 2+ ]. It is also permeable to Na + and K + . Large unitary conductances were measured in the presence of these cations. With its Ca 2+ permeability and modulation by [Ca 2+ ], MLN1 could play a major role in Ca 2+ transport regulating lysosomal exocytosis and potentially other phenomena related to the tra⁄cking of late endosomes and lysosomes. ß
Mucolipidosis type IV (MLIV) is a lysosomal storage disorder caused by mutations in the MCOLN1 gene, a member of the transient receptor potential (TRP) cation channel gene family. The encoded protein, transient receptor potential mucolipin-1 (TRPML1), has been localized to lysosomes and late endosomes but the pathogenic mechanism by which loss of TRPML1 leads to abnormal cellular storage and neuronal cell death is still poorly understood. Yeast two-hybrid and co-immunoprecipitation (coIP) experiments identified interactions between TRPML1 and Hsc70 as well as TRPML1 and Hsp40. Hsc70 and Hsp40 are members of a molecular chaperone complex required for protein transport into the lysosome during chaperone-mediated autophagy (CMA). To determine the functional relevance of this interaction, we compared fibroblasts from MLIV patients to those from sex- and age-matched controls and show a defect in CMA in response to serum withdrawal. This defect in CMA was subsequently confirmed in purified lysosomes isolated from control and MLIV fibroblasts. We further show that the amount of lysosomal-associated membrane protein type 2A (LAMP-2A) is reduced in lysosomal membranes of MLIV fibroblasts. As a result of decreased CMA, MLIV fibroblasts have increased levels of oxidized proteins compared to control fibroblasts. We hypothesize that TRPML1 may act as a docking site for intralysosomal Hsc70 (ly-Hsc70) allowing it to more efficiently pull in substrates for CMA. It is also possible that TRPML1 channel activity may be required for CMA. Understanding the role of TRPML1 in CMA will undoubtedly help to characterize the pathogenesis of MLIV.
A monoclonal antibody which blocks InsP(3)-induced Ca(2+) release from isolated endoplasmic reticulum was used to isolate a novel 4.0 kb cDNA from a human erythroleukaemia (HEL) cell cDNA expression library. A corresponding mRNA transcript of approx. 4.2 kb was present in all human cell lines and tissues examined, but cardiac and skeletal muscle had an additional transcript of 6.4 kb. The identification in GenBank(R) of homologous expressed sequence tags from many tissues and organisms suggests that the gene is ubiquitously expressed in higher eukaryotes. The gene was mapped to human chromosome 19p13.1. The cDNA predicts a 100 kDa protein, designated Ca(2+) homoeostasis endoplasmic reticulum protein (CHERP), with two putative transmembrane domains, multiple consensus phosphorylation sites, a polyglutamine tract of 12 repeats and regions of imperfect tryptophan and histadine octa- and nona-peptide repeats. In vitro translation of the full-length cDNA produced proteins of M(r) 128000 and 100000, corresponding to protein bands detected by Western blotting of many cell types. CHERP was co-localized in HEL cells with the InsP(3) receptor by two-colour immunofluorescence. Transfection of HEL cells with antisense cDNA led to an 80% decline in CHERP within 5 days of antisense induction, with markedly decreased intracellular Ca(2+) mobilization by thrombin, decreased DNA synthesis and growth arrest, indicating that the protein has an important function in Ca(2+) homoeostasis, growth and proliferation.
Individuals with a heterozygous mutation at the ataxia-telangiectasia mutated gene ( ATM ) have been reported to be predisposed to ischemic heart disease. This report examined for the first time the effect of a heterozygous ATM mutation ( ATM ؉ / ؊ ) on plasma lipid levels and atherosclerosis intensity using, and ATM ؉ / ؊ / ApoE ؊ / ؊ mice. Our data demonstrated that the plasma cholesterol and triglyceride levels in ATM ؉ / ؊ and ATM ؉ / ؊ / LDLR ؊ / ؊ mice were approximately the same as those in ATM ؉ / ؉ and ATM ؉ / ؉ / LDLR ؊ / ؊ control mice, respectively. In contrast, the plasma cholesterol level was significantly higher in ATM ؉ / ؊ / ApoE ؊ / ؊ mice than in ATM ؉ / ؉ / ApoE ؊ / ؊ control mice. In addition, the ATM ؉ / ؊ / ApoE ؊ / ؊ mice showed higher plasma apoB-48 levels, slower clearance for plasma apoB-48-carrying lipoproteins, and more advanced atherosclerotic lesions in the aorta compared with the ATM ؉ / ؉ / ApoE ؊ / ؊ mice. These novel results suggest that the product of ATM is involved in an apoE-independent pathway for catabolism of apoB-48-carrying remnants; therefore, superimposition of a heterozygous ATM mutation onto an ApoE deficiency background reduces the clearance of apoB-48-carrying lipoproteins from the blood circulation and promotes the formation of atherosclerosis. The product of the ataxia-telangiectasia mutated gene ( ATM ) has been reported to be a nuclear protein and involved in several signaling pathways, including DNA damage recognition, cell cycle control, and meiotic recombination (for review, see 1). It is now known that a fraction of ATM is also present in the cytoplasm and associated with vesicular structures such as peroxisomes (2). Ataxiatelangiectasia patients [i.e., those individuals carrying mutations at both ATM alleles ( ATM Ϫ / Ϫ )] express a variety of progressive clinical symptoms, such as cerebellar ataxia, telangiectasias, and a high incidence of cancer (for review, see 3). Cells obtained from ataxia-telangiectasia patients are more sensitive to ionizing radiation and show increased chromosomal aberrations compared with those obtained from normal subjects (4). Individuals with an ATM mutation in one allele ( ATM ϩ / Ϫ ) are spared most of the symptoms of the disease but are predisposed to cancer (5). A close review of the literature suggests that heterozygous ATM deficiency might also increase the risk of atherosclerosis-related cardiovascular diseases. For example, Swift and Chase (6) reported that the age-related mortality of heterozygous ATM carriers was increased markedly compared with the general population and that ischemic heart disease was one of the underlying causes for the early death of these individuals. Ataxia-telangiectasia patients reportedly have increased plasma cholesterol and triglyceride levels (7), which are the two major risk factors for atherosclerosis. However, ataxia-telangiectasia patients do not usually live past 20 or 30 years of age, and atherosclerosis has not been studied in these individuals.More recently, a mouse mode...
We recently discovered a novel gene on chromosome 19p13.1 and its product, an integral endoplasmic reticulum (ER) membrane protein, termed CHERP (calcium homoeostasis endoplasmic reticulum protein). A monoclonal antibody against its C-terminal domain inhibits Ins(1,4,5) P (3)-induced Ca(2+) release from ER membrane vesicles of many cell types, and an antisense-mediated knockdown of CHERP in human erythroleukemia (HEL) cells greatly impaired Ca(2+) mobilization by thrombin. In the present paper, we explore further CHERP's function in Jurkat T-lymphocytes. Confocal laser immunofluorescence microscopy showed that CHERP was co-localized with the Ins(1,4,5) P (3) receptor throughout the cytoplasmic and perinuclear region, as previously found in HEL cells. Transfection of Jurkat cells with a lac I-regulated mammalian expression vector containing CHERP antisense cDNA caused a knockdown of CHERP and impaired the rise of cytoplasmic Ca(2+) (measured by fura-2 acetoxymethyl ester fluorescence) caused by phytohaemagglutinin (PHA) and thrombin. A 50% fall of CHERP decreased the PHA-induced rise of the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)), but Ca(2+) influx was unaffected. Greater depletion of CHERP (>70%) did not affect the concentration of Ins(1,4,5) P (3) receptors, but diminished the rise of [Ca(2+)](i) in response to PHA to =30% of that in control cells, decreased Ca(2+) influx and slowed the initial rate of [Ca(2+)](i) rise caused by thapsigargin, an inhibitor of the sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase, suggesting there was also some deficit in ER Ca(2+) stores. In CHERP-depleted cells the Ca(2+)-dependent activation and translocation of the key transcription factor NFAT (nuclear factor of activated T-cells) from cytoplasm to nucleus was suppressed. Furthermore, cell proliferation was greatly slowed (as in HEL cells) along with a 60% decrease in cyclin D1, a key regulator of progression through the G(1) phase of the cell cycle. These findings provide further evidence that CHERP is an important component of the ER Ca(2+)-mobilizing system in cells, and its loss impairs Ca(2+)-dependent biochemical pathways and progression through the cell cycle.
Phospholipase modulators have been shown to affect the topology of lipid bilayers and the formation of tubulo-vesicular structures, but the specific endogenous phospholipases involved have yet to be identified. Here we show that TRPML1 (MLN1), a Ca 2+ -permeable channel contributes to membrane remodeling through a serine-lipase consensus domain, and thus represents a novel type of bifunctional protein. Remarkably, this serine lipase active site determines the ability of MLN1 to generate tubulo-vesicular extensions in mucolipin-1-expressing oocytes, human fibroblasts and model membrane vesicles. Our demonstration that MLN1 is involved in membrane remodeling and the formation of extensions suggests that it may play a role in the formation of cellular processes linked to the late endosome/lysosome (LE/L) pathway. MLN1 is absent or mutated in patients with mucolipidosis IV (MLIV), a lysosomal disorder with devastating neurological and other consequences. This study provides potential insight into the pathophysiology of MLIV.
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