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Protein-protein interactions between the testis brain RNA-binding protein and the transitional endoplasmic reticulum ATPase, a cytoskeletal gamma actin and Trax in male germ cells and the brain.

Numerous functions have been proposed for the testis brain RNA-binding protein (TB-RBP) and its human homologue, Translin, ranging from mRNA transport and translational regulation to DNA rearrangement and repair. To gain insight into the likely functions of this 26 kDa protein, immunoprecipitation was used to identify proteins that interact with TB-RBP in mouse cytosolic extracts. Three proteins, the transitional endoplasmic reticulum ATPase, a cytoskeletal gamma actin, and Trax, were specifically immunoprecipitated with an affinity-purified antibody to recombinant mouse TB-RBP. In vitro binding assays with recombinant proteins and EM immunocytochemistry confirm that TB-RBP interacts with the TER ATPase in vitro and in vivo. Confocal microscopy has demonstrated that TB-RBP colocalizes with actin in the cytoplasm of male germ cells. The immunoprecipitation of Trax with TB-RBP confirms a published report demonstrating protein interactions between the two proteins in a yeast two-hybrid assay. These data support the hypothesis that TB-RBP serves as a link in attaching specific mRNAs to cytoskeletal structures and suggests an involvement for the ubiquitously expressed TER ATPase in intracellular and/or intercellular mRNA transport.

Actins↗

Identification of TER94, an AAA ATPase protein, as a Bam-dependent component of the Drosophila fusome.

The Drosophila fusome is a germ cell-specific organelle assembled from membrane skeletal proteins and membranous vesicles. Mutational studies that have examined inactivating alleles of fusome proteins indicate that the organelle plays central roles in germ cell differentiation. Although mutations in genes encoding skeletal fusome components prevent proper cyst formation, mutations in the bag-of-marbles gene disrupt the assembly of membranous cisternae within the fusome and block cystoblast differentiation altogether. To understand the relationship between fusome cisternae and cystoblast differentiation, we have begun to identify other proteins in this network of fusome tubules. In this article we present evidence that the fly homologue of the transitional endoplasmic reticulum ATPase (TER94) is one such protein. The presence of TER94 suggests that the fusome cisternae grow by vesicle fusion and are a germ cell modification of endoplasmic reticulum. We also show that fusome association of TER94 is Bam-dependent, suggesting that cystoblast differentiation may be linked to fusome reticulum biogenesis.

Adenosine Triphosphatases↗

Leishmania chagasi T-cell antigens identified through a double library screen.

Control of human visceral leishmaniasis in regions where it is endemic is hampered in part by limited accessibility to medical care and emerging drug resistance. There is no available protective vaccine. Leishmania spp. protozoa express multiple antigens recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the cause of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T-cell antigens and T-dependent B-cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide by screening with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second-step screen for their ability to cause proliferation and gamma interferon responses in T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The corresponding antigens were derived from glutamine synthetase, a transitional endoplasmic reticulum ATPase, elongation factor 1gamma, kinesin K39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L. chagasi mounted both cellular and antibody responses to these proteins. Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines.

Amino Acid Sequence↗

Isolation and characterization of the principal ATPase associated with transitional endoplasmic reticulum of rat liver.

The transfer of membranes from the endoplasmic reticulum to the Golgi apparatus occurs via 50-70 nm transition vesicles which derive from part-rough, part-smooth transitional elements of the endoplasmic reticulum (TER). Vesicle budding from the TER is an ATP-dependent process both in vivo and in vitro. An ATPase with a monomer molecular weight of 100 kD by SDS-PAGE has been isolated from TER and designated as TER ATPase. The native TER ATPase has been characterized as a hexamer of six 100-kD subunits by gel filtration. The protein catalyzes the hydrolysis of [gamma 32-P]ATP and is phosphorylated in the presence of Mg2+. It is distinct from the classical transport ATPases based on pH optima, ion effects, and inhibitor specificity. Electron microscopy of negatively stained preparations revealed the TER ATPase to be a ring-shaped structure with six-fold rotational symmetry. A 19-amino acid sequence of TER ATPase having 84% identity with valosin-containing protein and 64% identity with a yeast cell-cycle control protein CDC48p was obtained. Anti-synthetic peptide antisera to a 15-amino acid portion of the sequence of TER ATPase recognized a 100-kD protein from TER. These antisera reduced the ATP-dependent cell-free formation of transition vesicles from isolated TER of rat liver. In a reconstituted membrane transfer system, TER ATPase antisera inhibited transfer of radiolabeled material from endoplasmic reticulum to Golgi apparatus, while preimmune sera did not. The results suggest that the TER ATPase is obligatorily involved in the ATP requirements for budding of transition vesicles from the TER. cDNA clones encoding TER ATPase were isolated by immunoscreening a rat liver cDNA library with the affinity-purified TER ATPase antibody. A computer search of deduced amino acid sequences revealed the cloned TER ATPase to be the rat equivalent of porcine valosin-containing protein, a member of a novel family of ATP binding, homo-oligomeric proteins including the N-ethylmaleimide-sensitive fusion protein.

Adenosine Triphosphatases↗

Multisystem Proteinopathy

CLINICAL CHARACTERISTICS: Multisystem proteinopathy (MSP) is a genetically heterogeneous, multisystem degenerative disorder characterized by adult-onset proximal and distal muscle weakness (clinically resembling a limb-girdle muscular dystrophy syndrome), early-onset Paget disease of bone (PDB), and premature frontotemporal dementia (FTD). Muscle weakness progresses to involve other limb and respiratory muscles. PDB involves focal areas of increased bone turnover that typically lead to spine and/or hip pain and localized enlargement and deformity of the long bones; pathologic fractures occur on occasion. Early stages of FTD are characterized by dysnomia, dyscalculia, comprehension deficits, and paraphasic errors, with minimal impairment of episodic memory; later stages are characterized by inability to speak, auditory comprehension deficits for even one-step commands, alexia, and agraphia. Mean age at diagnosis for muscle disease is 43 years, PDB is 41 years, and FTD is 56 years. Dilated cardiomyopathy, amyotrophic lateral sclerosis, and Parkinson disease are now known to be part of the spectrum of findings associated with MSP. DIAGNOSIS/TESTING: The diagnosis of MSP is established in a proband with typical clinical findings and a heterozygous pathogenic variant in VCP, HNRNPA1, HNRNPA2B1, or SQSTM1 identified by molecular genetic testing. MANAGEMENT: Treatment of manifestations: Weight control to avoid obesity; physical therapy and stretching exercises to promote mobility and prevent contractures; occupational therapy and mechanical aids (canes, walkers, orthotics, wheelchairs) as needed for ambulation/mobility; surgical intervention for foot deformity and scoliosis as needed; respiratory aids when indicated; assisted living arrangements for muscle weakness and/or dementia; bisphosphonates to relieve pain and disability from PDB; social and emotional support; education regarding safety precautions. Surveillance: Echocardiogram and electrocardiogram with repeat cardiac evaluation every two to three years or earlier if symptomatic; annual pulmonary function studies; sleep studies as clinically indicated; annual alkaline phosphatase measurement; skeletal imaging as indicated for evaluation of PDB; neurologic and neuropsychological assessment every two to three years or more frequently as needed; multidisciplinary monitoring for respiratory, cardiac, musculoskeletal, and cognitive decline. GENETIC COUNSELING: MSP is inherited in an autosomal dominant manner. Most individuals diagnosed with MSP have an affected parent. Estimates based largely on VCP-MSP suggest that approximately 5% of individuals have a de novo pathogenic variant. Each child of an individual with MSP has a 50% chance of inheriting the MSP-related pathogenic variant. Marked intrafamilial variability may be observed among heterozygous family members, including differences in age at onset, severity, rate of progression, and the specific combination of manifestations. Once the MSP-related pathogenic variant has been identified in an affected family member, predictive testing for at-risk family members and prenatal/preimplantation genetic testing are possible.

Inclusion Body Myopathy with Early-Onset Paget Dis↗

Tyrosine phosphorylation of p97 regulates transitional endoplasmic reticulum assembly in vitro.

The ATPase associated with different cellular activities family member p97, associated p47, and the t-SNARE syntaxin 5 are necessary for the cell-free reconstitution of transitional endoplasmic reticulum (tER) from starting low-density microsomes. Here, we report that membrane-associated tyrosine kinase and protein-tyrosine phosphatase (PTPase) activities regulate tER assembly by stabilizing (PTPase) or destabilizing (tyrosine kinase) p97 association with membranes. Incubation with the PTPase inhibitor bpV(phen) inhibited tER assembly coincident with the enhanced tyrosine phosphorylation of endogenous p97 and its release from membranes. By contrast, the tyrosine kinase inhibitor, genistein, promoted tER formation and prevented p97 dissociation from membranes while increasing p97 association with the t-SNARE syntaxin 5. Purification of the endogenous tyrosine kinase activity from low-density microsomes led to the identification of JAK-2, whereas PTPH1 was identified as the relevant PTPase. The p97 tyrosine phosphorylation state is proposed to coordinate the assembly of the tER as a regulatory step of the early secretory pathway.

Adenosine Triphosphatases↗

Gapped alignment of protein sequence motifs through Monte Carlo optimization of a hidden Markov model.

BACKGROUND: Certain protein families are highly conserved across distantly related organisms and belong to large and functionally diverse superfamilies. The patterns of conservation present in these protein sequences presumably are due to selective constraints maintaining important but unknown structural mechanisms with some constraints specific to each family and others shared by a larger subset or by the entire superfamily. To exploit these patterns as a source of functional information, we recently devised a statistically based approach called contrast hierarchical alignment and interaction network (CHAIN) analysis, which infers the strengths of various categories of selective constraints from co-conserved patterns in a multiple alignment. The power of this approach strongly depends on the quality of the multiple alignments, which thus motivated development of theoretical concepts and strategies to improve alignment of conserved motifs within large sets of distantly related sequences. RESULTS: Here we describe a hidden Markov model (HMM), an algebraic system, and Markov chain Monte Carlo (MCMC) sampling strategies for alignment of multiple sequence motifs. The MCMC sampling strategies are useful both for alignment optimization and for adjusting position specific background amino acid frequencies for alignment uncertainties. Associated statistical formulations provide an objective measure of alignment quality as well as automatic gap penalty optimization. Improved alignments obtained in this way are compared with PSI-BLAST based alignments within the context of CHAIN analysis of three protein families: Gialpha subunits, prolyl oligopeptidases, and transitional endoplasmic reticulum (p97) AAA+ ATPases. CONCLUSION: While not entirely replacing PSI-BLAST based alignments, which likewise may be optimized for CHAIN analysis using this approach, these motif-based methods often more accurately align very distantly related sequences and thus can provide a better measure of selective constraints. In some instances, these new approaches also provide a better understanding of family-specific constraints, as we illustrate for p97 ATPases. Programs implementing these procedures and supplementary information are available from the authors.

Amino Acid Motifs↗

Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly.

Although nuclear envelope (NE) assembly is known to require the GTPase Ran, the membrane fusion machinery involved is uncharacterized. NE assembly involves formation of a reticular network on chromatin, fusion of this network into a closed NE and subsequent expansion. Here we show that p97, an AAA-ATPase previously implicated in fusion of Golgi and transitional endoplasmic reticulum (ER) membranes together with the adaptor p47, has two discrete functions in NE assembly. Formation of a closed NE requires the p97-Ufd1-Npl4 complex, not previously implicated in membrane fusion. Subsequent NE growth involves a p97-p47 complex. This study provides the first insights into the molecular mechanisms and specificity of fusion events involved in NE formation.

Adenosine Triphosphatases↗

Effect of selenium-supplement on the calcium signaling in human endothelial cells.

Intracellular Ca2+ signaling controls many cellular functions. Understanding its regulation by selenoproteins is essential for understanding the role of selenoproteins in regulating cell functions. The activity of thioredoxin reductase (TrxR), thioredoxin (Trx) content, and the activity of glutathione peroxidase (GPx) in the human endothelial cells cultured in selenium-supplemented medium (refer as Se+ cells) was found 70%, 40%, and 20% higher, respectively than those in the cells cultured in normal medium (refer as Se0 cells). The intracellular Ca2+ signaling initiated by inositol 1,4,5-trisphosphate (IP3), histamine, thapsigargin (TG), carbonyl cyanide p-(tri-fluoromethoxy) phenyl-hydrazone (FCCP), and cyclosporin A (CsA) was investigated in both Se+ and Se0 cells. It was interestingly found that the higher activity of selenoproteins reduced the sensitivity of IP3 receptor to the IP3-triggered Ca2+ release from intracellular stores, but enhanced activation of the receptor-coupled phospholipase C in histamine-stimulated Se+ cells by showing much more generation of IP3 and higher elevation of cytosolic Ca2+. The higher selenoprotein activity also reduced susceptibility of the uniporter to the mitochondrial uncoupler, susceptibility of the permeability transition pore (PTP) to its inhibitor, and the vulnerability of endoplasmic reticulum (ER) Ca2+-ATPase to its inhibitor in selenium-supplementing cells. The results suggest that cell calcium signaling is subjected to thiol-redox regulation by selenoproteins.

Calcium↗

Protein transport into "complex" diatom plastids utilizes two different targeting signals.

The plastids found in diatoms and other chromophytic algae are completely enclosed by four membranes in contrast to chloroplasts of higher plants, which are surrounded by only two membranes. The bipartite targeting sequence of diatom nuclear-encoded plastid proteins contains an endoplasmic reticulum signal sequence and, based on sequence comparison, a transit peptide-like domain similar to that which targets proteins into the plastids of higher plants. By performing heterologous import experiments using the precursor of the gamma subunit of the chloroplast ATPase from the diatom Odontella sinensis we were able to show that protein import into diatom plastids is at least a two-step event. We demonstrate that the first step involves co-translational transport through endoplasmic reticulum membranes and that there is an additional targeting step which is similar to the import of precursor proteins into chloroplasts of higher plants and green algae indicating that the transit peptide-like domain of the diatom precursor is functionally equivalent to the respective targeting signal of higher plants. Our results suggest that the transit peptide depending targeting mechanism in plastids has apparently remained relatively unchanged over the course of evolution, with only the peptidase cleavage site significantly modified.

Algal Proteins↗

Bax and the mitochondrial permeability transition cooperate in the release of cytochrome c during endoplasmic reticulum-stress-induced apoptosis.

Endoplasmic reticulum (ER) stress induces apoptosis by mechanisms that are not fully clear. Here we show that ER stress induced by the Ca(2+)-ATPase inhibitor thapsigargin (THG) activates cytochrome c-dependent apoptosis through cooperation between Bax and the mitochondrial permeability transition (MPT) in human leukemic CEM cells. Pharmacological inhibition of the MPT as well as small interfering RNA (siRNA) knockdown of the MPT core component cyclophilin D blocked cytochrome c release and caspase-dependent apoptosis but did not prevent Bax activation, translocation or N-terminal exposure in mitochondria. siRNA knockdown of Bax also blocked THG-mediated cytochrome c release and apoptosis, but did not prevent MPT activation and resulted in caspase-independent cell death. Our results show that ER-stress-induced cell death involves a caspase and Bax-dependent pathway as well as a caspase-independent MPT-directed pathway.

Animals↗

Abnormal acidification of melanoma cells induces tyrosinase retention in the early secretory pathway.

In tyrosinase-positive amelanotic melanoma cells, inactive tyrosinase accumulates in the endoplasmic reticulum. Based on studies described here, we propose that aberrant vacuolar proton ATPase (V-ATPase)-mediated proton transport in melanoma cells disrupts tyrosinase trafficking through the secretory pathway. Amelanotic but not melanotic melanoma cells or normal melanocytes display elevated proton export as observed by the acidification of the extracellular medium and their ability to maintain neutral intracellular pH. Tyrosinase activity and transit through the Golgi were restored by either maintaining the melanoma cells in alkaline medium (pH 7.4-7.7) or by restricting glucose uptake. The translocation of tyrosinase out of the endoplasmic reticulum and the induction of cell pigmentation in the presence of the ionophore monensin or the specific V-ATPase inhibitors concanamycin A and bafilomycin A1 supported a role for V-ATPases in this process. Because it was previously shown that V-ATPase activity is increased in solid tumors in response to an acidified environment, the appearance of hypopigmented cells in tyrosinase-positive melanoma tumors may indicate the onset of enhanced glycolysis and extracellular acidification, conditions known to favor metastatic spread and resistance to weak base chemotherapeutic drugs.

Acids↗

Cyclosporin A inhibits inositol 1,4,5-trisphosphate-dependent Ca2+ signals by enhancing Ca2+ uptake into the endoplasmic reticulum and mitochondria.

Cytosolic Ca(2+) ([Ca(2+)](i)) oscillations may be generated by the inositol 1,4,5-trisphosphate receptor (IP(3)R) driven through cycles of activation/inactivation by local Ca(2+) feedback. Consequently, modulation of the local Ca(2+) gradients influences IP(3)R excitability as well as the duration and amplitude of the [Ca(2+)](i) oscillations. In the present work, we demonstrate that the immunosuppressant cyclosporin A (CSA) reduces the frequency of IP(3)-dependent [Ca(2+)](i) oscillations in intact hepatocytes, apparently by altering the local Ca(2+) gradients. Permeabilized cell experiments demonstrated that CSA lowers the apparent IP(3) sensitivity for Ca(2+) release from intracellular stores. These effects on IP(3)-dependent [Ca(2+)](i) signals could not be attributed to changes in calcineurin activity, altered ryanodine receptor function, or impaired Ca(2+) fluxes across the plasma membrane. However, CSA enhanced the removal of cytosolic Ca(2+) by sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), lowering basal and inter-spike [Ca(2+)](i). In addition, CSA stimulated a stable rise in the mitochondrial membrane potential (DeltaPsi(m)), presumably by inhibiting the mitochondrial permeability transition pore, and this was associated with increased Ca(2+) uptake and retention by the mitochondria during a rise in [Ca(2+)](i). We suggest that CSA suppresses local Ca(2+) feedback by enhancing mitochondrial and endoplasmic reticulum Ca(2+) uptake, these actions of CSA underlie the lower IP(3) sensitivity found in permeabilized cells and the impaired IP(3)-dependent [Ca(2+)](i) signals in intact cells. Thus, CSA binding proteins (cyclophilins) appear to fine tune agonist-induced [Ca(2+)](i) signals, which, in turn, may adjust the output of downstream Ca(2+)-sensitive pathways.

Animals↗

Mechanism of the calcium pump in the endoplasmic reticulum of liver: phosphoproteins as reaction intermediates.

Microsomal fractions, highly enriched with endoplasmic reticulum of rat and human liver exhibit Ca2+ uptake catalyzed by a Ca2+-pumping ATPase. The mechanism of Ca2+-translocation involves: (i) reversible Ca2+-dependent formation of an acyl-phosphoenzyme intermediate (Mr 116,000 to 118,000) with bound Ca2+, which in the reversed reaction can transphosphorylate its Pi to ADP to re-synthesize ATP; (ii) reversible transition of the ADP-reactive phosphoenzyme into an isomer without bound Ca2+, not further reactive to ADP; (iii) hydrolytic cleavage, stimulated by Mg2+, K+, and ATP of the ADP-unreactive phosphoenzyme with liberation of Pi. By analogy to a mechanism proposed for the Ca2+ pump of sarcoplasmic reticulum, the translocation of Ca2+ to and dissociation from the inner side of the membrane is suggested to occur by a conformational change, coupled with a decrease in Ca2+-affinity of the phosphoenzyme during its transition into the ADP-unreactive isomer. With CaATP as the effective substrate the reactions proceed normally but at a considerably slower rate.

Adenosine Triphosphate↗

Localization of a highly active pool of type II phosphatidylinositol 4-kinase in a p97/valosin-containing-protein-rich fraction of the endoplasmic reticulum.

Different phosphoinositides are synthesized in cell membranes in order to perform a variety of functions. One of the most abundant of these lipids is phosphatidylinositol (PI) 4-phosphate (PI4P), which is formed in human eukaryotes by type II and type III phosphatidylinositol 4-kinase (PI4K II and III) activities. PI4K II activity occurs in many different subcellular membranes, although no detailed analysis of the distribution of this activity has been reported. Using density gradient ultracentrifugation, we have previously found that in A431 cells the predominant PI4K activity arises from a type II alpha enzyme that is localized to a buoyant membrane fraction of unknown origin [Waugh, Lawson, Tan and Hsuan (1998) J. Biol. Chem. 273, 17115-17121]. We show here that these buoyant membranes contain an activated form of PI4K II alpha that can be separated from the bulk of the PI4K II alpha protein in A431 and COS-7 cells. Proteomic analysis revealed that the buoyant membrane fraction contains numerous endoplasmic reticulum (ER)-marker proteins, although it was separated from the bulk of the ER, ER-Golgi intermediate compartment, transitional ER, Golgi and other major subcellular membranes. Furthermore, the majority of the cytoplasmic valosin-containing protein (VCP), an AAA+ATPase implicated in various ER, transitional ER, Golgi and nuclear functions, was almost completely localized to the same buoyant membrane fraction. Co-localization of VCP and PI4K activity was confirmed by co-immunoprecipitation. These results suggest the previously unsuspected existence of an ER-related domain in which the bulk of the cellular PI4P synthesis and VCP are localized.

1-Phosphatidylinositol 4-Kinase↗

The sarcoplasmic reticulum Ca(2+)-ATPase, SERCA1a, contains endoplasmic reticulum targeting information.

The fast-twitch skeletal muscle Ca(2+)-ATPase isoenzyme, SERCA1a, is localized in chick skeletal myotubes to both the sarcoplasmic reticulum (SR) and to the nuclear envelope, an extension of the endoplasmic reticulum (ER). The ER labeling remained after cycloheximide treatment, indicating that it did not represent newly synthesized SERCA1a in transit to the SR. Expression of the cDNA encoding SERCA1a in cultured non-muscle cells led to the localization of the enzyme in the ER, as indicated by organelle morphology and the co-localization of SERCA1a with the endogenous ER luminal protein, BiP. Immunopurification analysis showed that SERCA1a was not bound to BiP, nor was any degradation apparent. Thus, the SR Ca(2+)-ATPase appears to contain ER targeting information.

Animals↗

Effects of brefeldin-A: potent inhibitor of intracellular protein transport on ultrastructure and resorptive function of cultured osteoclasts.

Brefeldin-A (BFA) is a specific and potent inhibitor of the intracellular transport of clathlin-uncoated transitional vesicles from the cisterns of rough-surfaced endoplasmic reticulum (RER) to the Golgi lamellae. This study was designed to clarify the effects of BFA on ultrastructure, subcellular localization of vacuolar-type H+-ATPase and a lysosomal cysteine proteinase, cathepsin K, in cultured osteoclasts and their resorptive function. H+-ATPase and cathepsin K are the most important enzymes for decalcification of apatite crystals and degradation of type-I collagen, respectively. In control cultures without BFA, osteoclasts were structurally characterized by the development of broad ruffled borders and clear zones, and formed many resorption lacunae in cocultured dentine slices. In BFA-treated cultures, osteoclasts lacked ruffled borders, and the cytoplasm was filled with regular-size and extremely large pale vacuoles over 2 microm in diameter, which were produced by fusion of adjacent vacuoles. BFA did not, however, inhibit clear zone formation and adhesion of osteoclasts to dentine slices. Resorption lacuna formation was markedly diminished by BFA treatment. Although H+-ATPase and cathepsin K were strongly expressed in osteoclast ruffled borders in control cultures, BFA treatment altered the subcellular localization and decreased the expression of these molecules. In BFA-treated cultures, H+-ATPase immunoreaction in osteoclasts was observed along the limiting membranes of some, but not all, regular-size pale vacuoles, but neither in extremely large vacuoles nor along the smooth plasma membranes facing the dentine slices. Similarly, cathepsin K was localized within lysosomes and some regular-size pale vacuoles, but its secretion toward the dentine slices through the ruffled borders was strongly inhibited by BFA treatment. These results suggest that 1.) formation of the osteoclast ruffled borders and their resorptive function are closely associated with the intracellular transport of these molecules from the RER cisterns and the Golgi lamellae to the ruffled borders, and 2.) both H+-ATPase and cathepsin K are selectively transported to the ruffled border membranes by pale vacuoles.

Animals↗