Tripeptidyl peptidase I, the late infantile neuronal ceroid lipofuscinosis gene product, initiates the lysosomal degradation of subunit c of ATP synthase

The specific accumulation of a hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of NCL (LINCL) is caused by a defect in the CLN2 gene product, tripeptidyl peptidase I (TPP-I). The data here show that TPP-I is involved in the initial degradation of subunit c in lysosomes and suggest that its absence leads directly to the lysosomal accumulation of subunit c. The inclusion of a specific inhibitor of TPP-I, Ala-Ala-Phe-chloromethylketone (AAF-CMK), in the culture medium of normal fibroblasts induced the lysosomal accumulation of subunit c. In an in vitro incubation experiment the addition of AAF-CMK to mitochondrial-lysosomal fractions from normal cells inhibited the proteolysis of subunit c, but not the b-subunit of ATP synthase. The use of two antibodies that recognize the aminoterminal and the middle portion of subunit c revealed that the subunit underwent aminoterminal proteolysis, when TPP-I, purified from rat spleen, was added to the mitochondrial fractions. The addition of both purified TPP-I and the soluble lysosomal fractions, which contain various proteinases, to the mitochondrial fractions resulted in rapid degradation of the entire molecule of subunit c, whereas the degradation of subunit c was markedly delayed through the specific inhibition of TPP-I in lysosomal extracts by AAF-CMK. The stable subunit c in the mitochondrial-lysosomal fractions from cells of a patient with LINCL was degraded on incubation with purified TPP-I. The presence of TPP-I led to the sequential cleavage of tripeptides from the N-terminus of the peptide corresponding to the amino terminal sequence of subunit c.     

New insight into lysosomal protein storage disease: Delayed catabolism of ATP synthase subunit c in batten disease

Subunit c is normally present as an inner mitochondrial membrane component of the Fo sector of the ATP synthase complex, but in the late infantile form of neuronal ceroid lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. Mechanism for specific accumulation of subunit c in lysosomes is not known. The rate of degradation of subunit c as measured by pulsechase and immunoprecipitation showed a marked delay of degradation in patients fibroblasts with late infantile form of NCL. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. Measurement of labeled subunit c in mitochondrial and lysosomal fractions showed that the accumulation of labeled subunit c in the mitochondrial fraction can be detected before lysosomal appearance of radioactive subunit c, suggesting that subunit c accumulated as a consequence of abnormal catabolism in the mitochondrion and is transferred to lysosomes, through an autophagic process. There were no large differences of various lysosomal protease activities between control and patient cells. In patient cells sucrose loading caused a marked shift of lysosomal density, but did not a shift of subunit c containing storage body. The biosynthetic rate of subunit c and mRNA levels for P1 and P2 genes that code for it were almost the same in both control and patient cells. These findings suggest that a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes.Special issue dedicated to Dr. Leon S. Wolfe.     

Specific Delay of Degradation of Mitochondrial ATP Synthase Subunit c in Late Infantile Neuronal Ceroid Lipofuscinosis (Batten Disease)

Abstract: Subunit c is normally present as an inner mitochondrial membrane component of the F₀ section of the ATP synthase complex, but in the late infantile form of neuronal ceroid lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. To explore the mechanism of storage of subunit c, the rates of degradation and synthesis of subunit c were measured in fibroblast cell types from controls and patients with the late infantile form of NCL. The radiolabel from subunit c decreased with time in control cells, whereas no apparent loss of radioactivity of subunit c was found in patients' cells. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. A combination of pulse-chase and subcellular fractionation analysis showed that a delay of intramitochondrial loss from prelabeled subunit c was seen in all diseased cells tested. Lysosomal appearance of labeled subunit c could be detected after chase for more than 1 week and its radioactivities were variable among diseased cell types. The biosynthetic rate of subunit c was almost the same in both control and patient cells. Northern blotting analyses showed that mRNAs for P1 and P2 genes had no significant difference in lengths and amounts between control and patient cells. Results suggest a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes. This is the first direct evidence to show a delay of subunit c degradation in the cells from the late infantile form of NCL.     

Specific storage of subunit c of mitochondrial ATP synthase in lysosomes of neuronal ceroid lipofuscinosis (Batten's disease).

Immunochemical studies demonstrated the specific accumulation of subunit c of mitochondrial ATP synthase in the brain homogenates of late infantile and juvenile forms of Batten's disease. It is not stored in the infantile form. Storage of subunit alpha of mitochondrial ATP synthase and cytochrome c oxidase subunit IV, an inner membrane protein of mitochondria was not detected in the brains. There was also no difference in the levels of cathepsin B between the two forms of Batten's disease and controls. In cultured skin fibroblasts subunit c accumulates in the late infantile form, whereas it does not in other lysosomal storage diseases. Crude mitochondrial lysosomal preparations of control fibroblasts were separated into high-density fractions rich in a lysosomal marker and low-density fractions rich in a mitochondrial marker on Percoll density gradients. Subunit c was mostly recovered in low-density mitochondrial fractions, but in cells from the late infantile disease a part of subunit c was recovered in the high-density lysosomal fractions. Immunolocalization studies demonstrated a dot-like staining of storage materials for subunit c in the cells from late infantile patients and the staining pattern of subunit c is similar to that of a lysosomal membrane marker, lgp120. Immunostaining failed to detect subunit c in control cells. These results indicate a specific accumulation of subunit c in lysosomes, and suggest that the two forms of Batten's disease are caused by a specific failure in the degradation of subunit c.     

A Lysosomal Proteinase, the Late Infantile Neuronal Ceroid Lipofuscinosis Gene (CLN2) Product, Is Essential for Degradation of a Hydrophobic Protein, the Subunit c of ATP Synthase

The specific accumulation of the hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of neuronal ceroid lipofuscinosis (LINCL) is caused by lysosomal proteolytic dysfunction. The defective gene in LINCL (CLN2 gene) has been identified recently. To elucidate the mechanism of lysosomal storage of subunit c, antibodies against the human CLN2 gene product (Cln2p) were prepared. Immunoblot analysis indicated that Cln2p is a 46-kDa protein in normal control skin fibroblasts and carrier heterozygote cells, whereas it was absent in cells from four patients with LINCL. RT-PCR analysis indicated the presence of mRNA for CLN2 in cells from the four different patients tested, suggesting a low efficiency of translation of mRNA or the production of the unstable translation products in these patient cells. Pulse-chase analysis showed that Cln2p was synthesized as a 67-kDa precursor and processed to a 46-kDa mature protein (t(1/2) = 1 h). Subcellular fractionation analysis indicated that Cln2p is localized with cathepsin B in the high-density lysosomal fractions. Confocal immunomicroscopic analysis also revealed that Cln2p is colocalized with a lysosomal soluble marker, cathepsin D. The immunodepletion of Cln2p from normal fibroblast extracts caused a loss in the degradative capacity of subunit c, but not the beta subunit of ATP synthase, suggesting that the absence of Cln2p provokes the lysosomal accumulation of subunit c.     

Degradation of short and long lived proteins in isolated rat liver lysosomes. Effects of pH, temperature, and proteolytic inhibitors

Most previous studies on inhibitors of lysosomal protein breakdown have been performed on isolated or cultured cells or on perfused organs. We have tested various inhibitors of proteolysis on lysosomes isolated from livers of rats injected with [14C]leucine 15 min (short labeling time) and 16 h (long labeling time) before killing. Intact lysosomes were incubated with different inhibitors (leupeptin, propylamine, E-64, pepstatin, and chloroquine) in increasing concentrations. None of these caused more than a 40-75% inhibition of proteolysis irrespective of labeling protocol. Chloroquine was the most effective inhibitor, followed by leupeptin, propylamine, E-64, and pepstatin. When lysosomes were incubated with various combinations of inhibitors, including a weak base and an enzyme inhibitor, a somewhat higher inhibition (86%) was obtained. To assess if lysosomes are active in the degradation of both short and long lived proteins, lysosomes were isolated from livers of rats labeled with [14C]leucine for various time intervals. The highest fractional proteolytic rates were seen for short lived proteins. If the recovery of the isolated lysosomes is taken into consideration, about 80% (short labeling time) and 90% (long labeling time) of the total proteolysis in the homogenate could be accounted for by lysosomes. Isolated Golgi, mitochondrial, and microsomal fractions displayed negligible proteolytic activities. The cytosol contributed one-fifth of the total protein breakdown of short lived proteins, whereas insignificant proteolysis was recovered in the cytosolic fraction following long time labeling. Accordingly, we propose that 1) lysosomal inhibitors do not completely suppress proteolysis in isolated lysosomes and that 2) both short and long lived proteins are degraded in lysosomes.     

A Cytotoxic Type III Secretion Effector of Vibrio parahaemolyticus Targets Vacuolar H+-ATPase Subunit c and Ruptures Host Cell Lysosomes

Vibrio parahaemolyticus is one of the human pathogenic vibrios. During the infection of mammalian cells, this pathogen exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1). VepA, an effector protein secreted via the T3SS1, plays a major role in the T3SS1-dependent cytotoxicity of V. parahaemolyticus. However, the mechanism by which VepA is involved in T3SS1-dependent cytotoxicity is unknown. Here, we found that protein transfection of VepA into HeLa cells resulted in cell death, indicating that VepA alone is cytotoxic. The ectopic expression of VepA in yeast Saccharomyces cerevisiae interferes with yeast growth, indicating that VepA is also toxic in yeast. A yeast genome-wide screen identified the yeast gene VMA3 as essential for the growth inhibition of yeast by VepA. Although VMA3 encodes subunit c of the vacuolar H⁺-ATPase (V-ATPase), the toxicity of VepA was independent of the function of V-ATPases. In HeLa cells, knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity. We also demonstrated that VepA interacted with V-ATPase subunit c, whereas a carboxyl-terminally truncated mutant of VepA (VepAΔC), which does not show toxicity, did not. During infection, lysosomal contents leaked into the cytosol, revealing that lysosomal membrane permeabilization occurred prior to cell lysis. In a cell-free system, VepA was sufficient to induce the release of cathepsin D from isolated lysosomes. Therefore, our data suggest that the bacterial effector VepA targets subunit c of V-ATPase and induces the rupture of host cell lysosomes and subsequent cell death.     

Abnormalities of lysosomes in human diploid fibroblasts from patients with Farber's disease

An accumulation of ceramide associated with the deficiency of acid ceramidase has been demonstrated in cultured diploid skin fibroblasts from a patient with Farber's disease. We extend this observation to investigate the lysosomal localization of accumulated ceramide and the abnormalities of lysosomes caused by this ceramide accumulation in Farber's diseased fibroblasts. We have found that the lysosomal fraction isolated from Farber's diseased fibroblasts by a subcellular fractionation procedure is markedly low in density compared with that of normal fibroblasts and is separated from other subcellular organellers. Ultrastructural studies of the isolated lysosomal fraction from Farber's diseased fibroblasts showed in mixed population of intact and swollen vesicles with a lysosomal appearance. Examination under high magnification clearly demonstrated lysosomal inclusions which contain lamellar and curvilinear membranes and resembled those seen in the intact fibroblasts. Subcellular localization of Farber's fibroblasts showed that the accumulated [³H]ceramide from the culture medium was predominantly localized in the lysosomal fraction with a markedly low density and very little was found to be associated with other cellular membranes. Our finding that ceramide is accumulated in the lysosomal fraction of Farber's fibroblasts and that these cells also show membranous inclusions strongly suggests that the accumulation of ceramide is directly involved in the formation of lysosomal inclusions.     

Characterization of lipid-linked oligosaccharide accumulation in mouse models of Batten disease

The neuronal ceroid lipofuscinoses (NCLs, also known collectively as Batten disease) are a group of lysosomal storage disorders characterized by the accumulation of autofluorescent storage material in the brain and other tissues. A number of genes underlying various forms of NCL have been cloned, but the basis for the neurodegeneration in any of these is unknown. High levels of dolichol pyrophosphoryl oligosaccharides have previously been demonstrated in brain tissue from several NCL patients, but the specificity of the effect for the NCLs has been unclear. In the present study, we examine eight mouse models of lysosomal storage disorders by modern FACE and found striking lipid-linked oligosaccharide (LLO) accumulation in NCL mouse models (especially CLN1, CLN6, and CLN8 knockout or mutant mice) but not in several other lysosomal storage disorders affecting the brain. Using a mouse model of the most severe form of NCL (the PPT1 knockout mouse), we show that accumulated LLOs are not the result of a defect in LLO synthesis, extension, or transfer but rather are catabolic intermediates derived from LLO degradation. LLOs are enriched about 60-fold in the autofluorescent storage material purified from PPT1 knockoutmouse brain but comprise only 0.3% of the autofluorescent storage material by mass. The accumulation of LLOs is postulated to result from inhibition of late stages of lysosomal degradation of autophagosomes, which may be enriched in these metabolic precursors.     

Proteolytic degradation of glutamate decarboxylase mediates disinhibition of hippocampal CA3 pyramidal cells in cathepsin D-deficient mice

Although of clinical importance, little is known about the mechanism of seizure in neuronal ceroid lipofuscinosis (NCL). In the present study, we have attempted to elucidate the mechanism underlying the seizure of cathepsin D-deficient (CD-/-) mice that show a novel type of lysosomal storage disease with a phenotype resembling late infantile NCL. In hippocampal slices prepared from CD-/- mice at post-natal day (P)24, spontaneous burst discharges were recorded from CA3 pyramidal cells. At P24, the mean amplitude of IPSPs after stimulation of the mossy fibres was significantly smaller than that of wild-type mice, which was substantiated by the decreased level of gamma-aminobutyric acid (GABA) contents in the hippocampus measured by high-performance liquid chromatography (HPLC). At this stage, activated microglia were found to accumulate in the pyramidal cell layer of the hippocampal CA3 subfield of CD-/- mice. However, there was no significant change in the numerical density of GABAergic interneurons in the CA3 subfield of CD-/- mice at P24, estimated by counting the number of glutamate decarboxylase (GAD) 67-immunoreactive somata. In the hippocampus and the cortex of CD-/- mice at P24, some GABAergic interneurons displayed extremely high somatic granular immunoreactivites for GAD67, suggesting the lysosomal accumulation of GAD67. GAD67 levels in axon terminals abutting on to perisomatic regions of hippocampal CA3 pyramidal cells was not significantly changed in CD-/- mice even at P24, whereas the total protein levels of GAD67 in both the hippocampus and the cortex of CD-/- mice after P24 were significantly decreased as a result of degradation. Furthermore, the recombinant human GAD65/67 was rapidly digested by the lysosomal fraction prepared from the whole brain of wild-type and CD-/- mice. These observations strongly suggest that the reduction of GABA contents, presumably because of lysosomal degradation of GAD67 and lysosomal accumulation of its degraded forms, are responsible for the dysfunction of GABAergic interneurons in the hippocampal CA3 subfield of CD-/- mice.     

Scrapie protein degradation by cysteine proteases in CD11c+ dendritic cells and GT1-1 neuronal cells

Dendritic cells (DC) of the CD11c(+) myeloid phenotype have been implicated in the spread of scrapie in the host. Previously, we have shown that CD11c(+) DC can cause a rapid degradation of proteinase K-resistant prion proteins (PrP(Sc)) in vitro, indicating a possible role of these cells in the clearance of PrP(Sc). To determine the mechanisms of PrP(Sc) degradation, CD11c(+) DC that had been exposed to PrP(Sc) derived from a neuronal cell line (GT1-1) infected with scrapie (ScGT1-1) were treated with a battery of protease inhibitors. Following treatment with the cysteine protease inhibitors (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane (E-64c), its ethyl ester (E-64d), and leupeptin, the degradation of PrP(Sc) was inhibited, while inhibitors of serine and aspartic and metalloproteases (aprotinin, pepstatin, and phosphoramidon) had no effect. An endogenous degradation of PrP(Sc) in ScGT1-1 cells was revealed by inhibiting the expression of cellular PrP (PrP(C)) by RNA interference, and this degradation could also be inhibited by the cysteine protease inhibitors. Our data show that PrP(Sc) is proteolytically cleaved preferentially by cysteine proteases in both CD11c(+) DC and ScGT1-1 cells and that the degradation of PrP(Sc) by proteases is different from that of PrP(C). Interference by protease inhibitors with DC-induced processing of PrP(Sc) has the potential to modify prion spread, clearance, and immunization in a host.     

Altered Mitochondrial Function in Canine Ceroid-Lipofuscinosis

The neuronal ceroid-lipofuscinoses (NCL) are a group of autosomal recessively inherited neurodegenerative disorders characterized by progressive dementia, neuronal atrophy, and premature death. The late infantile and juvenile types of NCL show massive accumulation of mitochondrial ATP synthase subunit c protein in both mitochondria and lysosomes. The specific accumulation of this mitochondrial protein suggests that mitochondrial function may be impaired in the NCL diseases. Therefore, a study was conducted to determine whether oxidative phosphorylation is altered in liver mitochondria from English setters with NCL, an animal model in which there is also massive accumulation of the subunit c protein. The ADP/O ratios were significantly depressed in affected and carrier dogs, suggesting that the disease mutation led to a partial uncoupling of oxidative phosphorylation. On the other hand, ADP-stimulated respiration rates were higher than normal in both carriers and affected dogs. The increased respiration rates were highest in the carriers, and may reflect a compensatory response to the reduced efficiency of oxidative phosphorylation. Accompanying the increased respiration rates were elevations in mitochondrial ADP content with the elevation being greater in the carriers than in the affected dogs. This suggests that the increased respiration rates may be due, at least in part, to enhanced ADP uptake by the mitochondria. In the carriers, the enhanced respiration rate may be sufficient to offset the reduced efficiency of oxidative phosphorylation. In the affected animals, which had lower respiration rates than the carriers, the enhanced respiration rates may not be sufficient to offset the reduced efficiency of oxidative phosphorylation. Impaired mitochondrial function may therefore contribute to the disease pathology.     

Role of lysosomal acid ceramidase in the metabolism of ceramide in human skin fibroblasts

A 2.8-fold accumulation of ceramide was demonstrated in cultured skin ftbroblasts from a patient with Farber's disease, an inborn error of metabolism in which acid ceramidase activity is deficient. To investigate the role of acid ceramidase in the metabolism of ceramide in fibroblasts, we have investigated the lysosomal degradation of ceramide that was taken up by fibroblasts from an exogenous lipid suspension. Fluorescent 4-nitrobenz-2-oxa-1,3-diazole-7-aminododecanoyl-sphingosine (NBD-ceramide) from an exogenous ceramide suspension was incorporated into the intracellular structures of fibroblasts at 37 °C. Study of the cellular uptake of exogenous [³H]oleylsphingosine showed that the rate of ceramide accumulation was nearly identical in Farber's disease and normal fibroblasts. The deficiency of acid ceramidase in Farber's fibroblasts resulted in the decrease of cellular degradation and uptake of ceramide and the increase of retention time of ceramide in these diseased cells. Studies of subcellular fractionation of these fibroblasts showed that the accumulated ceramide was located in the lysosomal fraction. As a result, the density of the lysosomal fraction of Farber's fibroblasts was found to be less than that of controls. These results suggest the defect of cellular metabolism in this inherited disease is located within the lysosome.     

A missense mutation P136L in the arylsulfatase A gene causes instability and loss of activity of the mutant enzyme

Metachromatic leukodystrophy is a lysosomal storage disease caused by deficiency of arylsulfatase A. Sequencing of the arylsulfatase A genes of an Ashkenazi Jewish patient suffering from the severe late infantile form of the disease revealed a point mutation in exon 2 causing proline 136 to be substituted by leucine. The patient was homozygous for this mutation. Studies on Ltk^- cells stably expressing the mutant enzyme show that the mutation causes complete loss of enzyme activity and rapid degradation in an early biosynthetic compartment.     

The sequence of the major protein stored in ovine ceroid lipofuscinosis is identical with that of the dicyclohexylcarbodiimide-reactive proteolipid of mitochondrial ATP synthase.

The ceroid lipofuscinoses are a group of neurodegenerative lysosomal storage diseases of children and animals that are recessively inherited. In diseased individuals fluorescent storage bodies accumulate in a wide variety of cells, including neurons. Previous studies of these bodies isolated from tissues of affected sheep confirmed that the storage occurs in lysosomes, and showed that the storage body is mostly made of a single protein with an apparent molecular mass of 3500 Da with an N-terminal amino acid sequence that is the same as residues 1-40 of the c-subunit (or dicyclohexylcarbodi-imide-reactive proteolipid) of mitochondrial ATP synthase. In the present work we have shown by direct analysis that the stored protein is identical in sequence with the entire c-subunit of mitochondrial ATP synthase, a very hydrophobic protein of 75 amino acid residues. As far as can be detected by the Edman degradation, the stored protein appears not to have been subject to any post-translational modification other than the correct removal of the mitochondrial import sequences that have been shown in other experiments to be present at the N-terminal of its two different precursors. No other protein accumulates in the storage bodies to any significant extent. Taken with studies of the cDNAs for the c-subunit in normal and diseased sheep, these results indicate that the material that is stored in lysosomes of diseased animals has probably entered mitochondria and has been subjected to the proteolytic processing that is associated with mitochondrial import. This implies that the defect that leads to the lysosomal accumulation concerns the degradative pathway of the c-subunit of ATP synthase. An alternative, but less likely, hypothesis is that for some unknown reason the precursors of subunit c are being directly mis-targeted to lysosomes, where they become processed to yield a protein identical with the protein that is normally found in the mitochondrial ATP synthase assembly, and which then accumulates.     

Biogenesis of Lysosome-related Organelles Complex-1 Subunit 1 (BLOS1) Interacts with Sorting Nexin 2 and the Endosomal Sorting Complex Required for Transport-I (ESCRT-I) Component TSG101 to Mediate the Sorting of Epidermal Growth Factor Receptor into Endosomal Compartments

Biogenesis of lysosome-related organelles complex-1 (BLOC-1) is a component of the molecular machinery required for the biogenesis of specialized organelles and lysosomal targeting of cargoes via the endosomal to lysosomal trafficking pathway. BLOS1, one subunit of BLOC-1, is implicated in lysosomal trafficking of membrane proteins. We found that the degradation and trafficking of epidermal growth factor receptor (EGFR) were delayed in BLOS1 knockdown cells, which were rescued through BLOS1 overexpression. A key feature to the delayed EGFR degradation is the accumulation of endolysosomes in BLOS1 knockdown cells or BLOS1 knock-out mouse embryonic fibroblasts. BLOS1 interacted with SNX2 (a retromer subunit) and TSG101 (an endosomal sorting complex required for transport subunit-I) to mediate EGFR lysosomal trafficking. These results suggest that coordination of the endolysosomal trafficking proteins is important for proper targeting of EGFR to lysosomes.     

Targeting of asialofetuin sugar chain-bearing liposomes to liver lysosomes

Specific direction of liposomes bearing an asialofetuin sugar chain (AFSC) to liver parenchymal cells was examined both in vivo and in vitro. The AFSC-bearing liposomes were preferentially recovered in the liver within several minutes after an intravenous injection into mice and were found to be predominantly localized in mitochondrial-lysosomal fraction. The massive distribution of the AFSC-liposomes in this fraction was also confirmed by using a lysosomal protease inhibitor, E-64-d. In isolated rat hepatocytes, the uptake of AFSC-liposomes was increased 2-3-fold as compared with the control liposomes without AFSC. Thus liposomes bearing AFSC would be useful to target enzymes to liver lysosomes.     

Defective endoplasmic reticulum-resident membrane protein CLN6 affects lysosomal degradation of endocytosed arylsulfatase A

Variant late infantile neuronal ceroid lipofuscinosis, a lysosomal storage disorder characterized by progressive mental deterioration and blindness, is caused by mutations in a polytopic membrane protein (CLN6) with unknown intracellular localization and function. In this study, transient transfection of BHK21 cells with CLN6 cDNA and immunoblot analysis using peptide-specific CLN6 antibodies demonstrated the expression of a approximately 27-kDa protein that does not undergo proteolytic processing. Cross-linking experiments revealed the presence of CLN6 dimers. Using double immunofluorescence microscopy, epitope-tagged CLN6 was shown to be retained in the endoplasmic reticulum (ER) with no colocalization with the cis-Golgi or lysosomal markers. The translocation into the ER and proper folding were confirmed by the N-linked glycosylation of a mutant CLN6 polypeptide. Pulse-chase labeling of fibroblasts from CLN6 patients and from sheep (OCL6) and mouse (nclf) models of the disease followed by immunoprecipitation of cathepsin D indicated that neither the synthesis, sorting nor the proteolytic processing of this lysosomal enzyme was affected in CLN6-defective cells. However, the degradation of the endocytosed index protein arylsulfatase A was strongly reduced in all of the mutant CLN6 cell lines compared with controls. These data suggest that defects in the ER-resident CLN6 protein lead to lysosomal dysfunctions, which may result in lysosomal accumulation of storage material.     

Receptor-mediated introduction of pepstatin-asialofetuin conjugate into lysosomes of rat hepatocytes

Pepstatin was linked through a carboxyl group to asialofetuin (PS-ASF). An analysis by separation of hepatocytes from nonparenchymal cells showed that PS-ASF was taken up by hepatocytes, following intravenous injection into rats. After the injection of PS-ASF, pepstatin concentration in the liver reached a maximum at 2 h and then decreased. In an analysis by differential centrifugation of the liver homogenate from rats injected with PS-ASF, pepstatin showed a lysosomal type subcellular distribution pattern. Isolation studies of tritosomes clearly demonstrated the exclusive accumulation of pepstatin within the lysosomes of livers from rats given PS-ASF (at 2 h after administration). Pepstatin contained in tritosomes was in a free form, as determined by column chromatography of Sephadex G-15. The activity of cathepsin D in the livers was markedly inhibited in rats given PS-ASF. However, the treatment of rats with PS-ASF had no effect on the hepatic lysosomal degradation of endocytosed FITC-labeled asialofetuin (FITC-ASF). Introduction of PS-ASF into the hepatocytes was followed by the immediate and time-dependent excretion of free pepstatin into the bile. Quantification of pepstatin excreted into the bile revealed that the biliary excretion route can account for the disappearance of pepstatin from the liver.     

Subunit A of calmodulin-dependent protein phosphatase requires Mn2+ for activity

Bovine brain calmodulin-dependent protein phosphatase comprises a catalytic subunit A (Mr 60,000) and a regulatory subunit B (Mr 19,000). The native enzyme was active with Ca²⁺ or Mn²⁺. Upon resolution into its subunits in 6 M urea and 15 mM EDTA, subunit A was active with Mn²⁺; Co²⁺ and Ni²⁺ partially substituted for Mn²⁺, but Ca²⁺, Mg²⁺ and Zn²⁺ were ineffective. The stimulating effect of Mn²⁺ was not easily reversed by EGTA. Like the native phosphatase, subunit A was markedly stimulated by calmodulin or by controlled trypsinization. Unlike the native enzyme, however, trypsinized subunit A still required Mn²⁺ for activity. These findings provide evidence that the catalytic subunit of phosphatase may be a metallo (possibly Mn²⁺) enzyme.