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PubMed1982

Sugar transport by the bacterial phosphotransferase system. Nanosecond fluorescence studies of the phosphocarrier protein (HPr) labeled at the NH2-terminal methionine.

HPr is a low molecular weight, phosphocarrier protein of the Salmonella typhimurium phosphoenolpyruvate:glycose phosphotransferase system (PTS). This protein was alkylated with the fluorescent reagent (N-iodoacetylaminoethyl)-5-naphthylamino-1-sulfonate under conditions which favor alkylation of the thioether linkage in methionine residues (Link, T. P. and Stark, G. R. (1968) J. Biol. Chem. 243, 1082-1088) to give the corresponding sulfonium derivatives. The isolated fluorescent protein (95-100% pure) was as active as native HPr both as a phosphoryl acceptor protein (phosphoenolpyruvate and Enzyme I of the PTS), and as a phosphocarrier protein in the phosphorylation of methyl alpha-glucoside by the complete PTS. The fluorescent label was shown to be predominantly, possibly exclusively, at the NH2-terminal methionine residue. The decay of the fluorescence intensity could be described in terms of a biexponential function with the time constants tau 1 approximately 7 ns, tau 2 approximately 15 ns, and a ratio of alpha 2/alpha 1 approximately 3 for the pre-exponential factors. The decay of the fluorescence emission anisotropy was found to be consistent with some internal motion of the probe, in addition to the rotation of the protein conjugate as a whole.

Bacterial Proteins↗

PubMed1982

Sugar transport by the bacterial phosphotransferase system. Regulation of other transport systems (lactose and melibiose).

The role of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in the phenomenon of inducer exclusion was examined in whole cells of Salmonella typhimurium which carried the genes of the Escherichia coli lactose operon on an episome. In the presence of the PTS substrate methyl alpha-D-glucopyranoside, the extent of accumulation of the lactose analog methyl beta-D-thiogalactopyranoside was reduced. A strain carrying a mutation in the gene for Enzyme I was hypersensitive to the PTS effect, while a crr mutant strain was completely resistant. Influx, efflux, and exchange of galactosides via the lactose "permease" were inhibited by methyl alpha-glucoside. This inhibition occurred in the presence of metabolic energy poisons, and therefore does not involve either the generation of metabolic energy or energy-coupling to the lactose transport system. When the cellular content of the lactose permease was increased by induction with isopropyl beta-D-thiogalactopyranoside, cells gradually became less sensitive to inducer exclusion. The extent of inhibition of methyl beta-thiogalactoside accumulation by methyl alpha-glucoside was shown to be dependent on the relative cellular content of the PTS and lactose system. The data were consistent with an hypothesis involving partial inactivation of galactoside transport due to interaction between a component of the PTS and the lactose permease. By examination of the effects of the PTS and lactose uptake and melibiose permease-mediated uptake of methyl beta-thiogalactoside, it was further shown that the manner in which inducer exclusion is expressed is independent on the routes available to the non-PTS sugar for exit from the cell.

Biological Transport↗

PubMed1994

Retrovirus-mediated transfer of a hygromycin phosphotransferase-thymidine kinase fusion gene into human CD34+ bone marrow cells.

Retrovirus-mediated gene transfer into human hematopoietic stem cells has been proposed as a means of therapy for various inherited diseases and as a method of gene marking. The transduction efficiency of an amphotropic retroviral vector (PA317/HyTK) containing a hygromycin phosphotransferase-thymidine kinase fusion gene was examined with human CD34+ bone marrow cells in the presence of interleukin-3 (IL-3), interleukin-6 (IL-6), and stem cell factor. Transduction efficiencies determined from the ability of transduced granulocyte-macrophage colony forming units (CFU-GM) to grow in hygromycin B and from polymerase chain reaction analysis of individual transduced CFU-GM growing in the presence of hygromycin B were 0.3-3.0% (mean +/- S.D., 1.1 +/- 0.9%) and 0.1-1.2% (mean +/- S.D., 0.5 +/- 0.4%), respectively. Ganciclovir at a dose of approximately 1 microM reduced the number of CFU-GM derived from vector-infected CD34+ cells by 50%. These findings demonstrate that human hematopoietic stem cells infected with this retroviral vector are susceptible to ganciclovir, offering the potential to control transduced gene expression in vivo.

Antigens, CD↗

PubMed1976

Sugar transport. 2nducer exclusion and regulation of the melibiose, maltose, glycerol, and lactose transport systems by the phosphoenolpyruvate:sugar phosphotransferase system.

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) can repress the synthesis of certain catabolic enzyme systems in Salmonelly typhimurium and Escherichia coli. The present studies are concerned with an explanation of PTS-mediated repression by studying induction of the catabolic systems required for utilization of the non-PTS sugars glycerol, maltose, melibiose, and lactose. Repression of synthesis of these enzyme systems by various concentrations of PTS sugars was studied in wild type cells, in pts mutants, and in pts crr double mutants described in the accompanying reports (Saier, M. H., Jr., Simoni, R. D., and Roseman, S (1976) J. Biol. Chem. 251, 6584-6597: Saier, M. H., Jr., and Roseman, S. (1976) J. Biol. Chem., 6598-6605). The extent of repression was found to correlate with the degree of inhibition of uptake of the inducing non-PTS sugars. In both uninduced cells and cells fully induced for the respective transport system listed above, PTS sugars inhibited these transport systems. With both induced and uninduced cell types pts mutants were much more sensitive than wild type cells, while pts crr double mutants were completely resistant to the effects of the PTS sugars. Kinetic studies with the fully induced cells indicated that inhibition of transport by PTS sugars was reversible, that it affected the Vmax rather than the Km of entry, and that PTS sugars did not inhibit the efflux of the non-PTS sugars. These and other results indicate that inhibition did not result from competition between the PTS and non-PTS sugars for the transport systems, nor to competition for metabolic energy. Inhibition appears to require interaction of the PTS sugar with its membrane-bound Enzyme II complex, but whether concomitant uptake and phosphorylation of trace quantities of the PTS sugars is also required remains to be determined.

Biological Transport, Active↗

PubMedDate not supplied

[Use of the hygromycin phosphotransferase gene as the dominant selective marker for Chlamydomonas reinhardtii transformation].

The hygromycin phosphotransferase gene (hpt) from E. coli under the control of the SV40 early promoter was used as a dominant selectable marker for transformation of Chlamydomonas reinhardtii. Cells were transformed by electroporation (pulse length, 2 ms, field strength, 1 kV/cm). The culture growth phase was a crucial parameter for transformation (optimal density approximately 10(6) cells/ml). It was possible to obtain approximately 10(3) Hyg-resistant colonies under these conditions. Foreign DNA integrated into the Chlamydomonas genome was maintained for at least 8 months but the Hyg-resistant phenotype of the transformed clones was unstable. The frequency of codon usage in the hpt gene was compared with the one in Chlamydomonas nuclear genes. It is supposed that highly biased codon usage in Chlamydomonas does not preclude expression. Advantages of this selection system for studying Chlamydomonas transformation by heterologous genes are discussed.

Animals↗

PubMed1994

Sugar transport by the bacterial phosphotransferase system. Characterization of the Escherichia coli enzyme I monomer/dimer equilibrium by fluorescence anisotropy.

Enzyme I (EI), the first protein of the bacterial phosphotransferase system (PTS), exists in a monomer/dimer (M/D) equilibrium. We have proposed that the two species are functionally different and that their interconversion may regulate sugar transport via the PTS. The C-terminal Cys of Escherichia coli EI was reacted with pyrene maleimide (Han, M. K., Roseman, S., and Brand, L. (1990) J. Biol. Chem. 265, 1985-1995), and the pyrene conjugate used to characterize the M/D equilibrium by fluorescence anisotropy. The properties of unlabeled and pyrene-labeled EI are indistinguishable. Values for the apparent association constant, K'eq, and the steady-state anisotropy of the monomer and the dimer were obtained under a variety of conditions. K'eq increases 23-fold, from 0.45 x 10(5) to 10.7 x 10(5) M-1, as the temperature increases from 6 to 30 degrees C; the association appears to be entropically driven. Under all conditions tested, the K'eq for phospho-EI is 6-12-fold less than for dephospho-EI. For phospho-EI, PEP and Mg2+ induce a 240-fold increase of K'eq when both ligands are present. Based on these data, EI was preincubated under conditions that change K'eq, and the initial activities of the different species were determined at 37 degrees C in a PTS sugar phosphorylation assay with PEP as the phosphoryl donor. The initial rate depends on the M/D ratio; it is maximal when EI is 100% dimer, and zero when EI is 100% monomer. In the latter case, the rate gradually increases in the assay mixture. The results have important implications for how the PTS regulates sugar transport and other physiological phenomena.

Biological Transport↗

PubMed1994

Sugar transport by the bacterial phosphotransferase system. Characterization of the Escherichia coli enzyme I monomer/dimer transition kinetics by fluorescence anisotropy.

Enzyme I of the bacterial phosphoenolpyruvate: glycose phosphotransferase system (PTS) exists in a monomer/dimer (M/D) equilibrium. These two forms are functionally different, and their interconversion may be a means of regulating the PTS. The M/D equilibrium was studied by fluorescence anisotropy of a pyrene derivative (Chauvin, F., Brand, L., and Roseman, S. (1994) J. Biol. Chem. 269, 20263-20269). In this paper, the kinetics of the transition is investigated. The following apparent rate constants were found for the M/D transition of phospho-Enzyme I in the presence of Mg2+ and PEP at 6 degrees C: k*A = 3.4 x 10(3) M-1 s-1 and k*D = 1.04 x 10(3) s-1. The association rate is especially slow, 2-3 orders of magnitude slower than the average dimerization rate determined for other proteins. Furthermore, the rate of quaternary structure changes matches that of enzymatic activity changes, as well as that of tertiary structure changes (Chauvin, F., Toptygin, D., Roseman, S., and Brand, L. (1992) Biophys. Chem. 44, 163-173). Finally, the effect of two ligands is shown; PEP increases the relaxation rate by 3-fold at 23 degrees C, and Mg2+ addition causes a 4-fold increase in the relaxation rate.

Biological Transport↗

PubMed1994

Inhibition of the phosphoenolpyruvate:lactose phosphotransferase system and activation of a cytoplasmic sugar-phosphate phosphatase in Lactococcus lactis by ATP-dependent metabolite-activated phosphorylation of serine 46 in the phosphocarrier protein HPr.

Lactococcus lactis takes up lactose and the nonmetabolizable lactose analogue, thiomethyl-beta-galactoside (TMG), via the phosphoenolpyruvate:sugar phosphotransferase system (PTS) which couples sugar transport to sugar phosphorylation. Earlier studies had shown that TMG-phosphate, previously accumulated in L. lactis cells, is rapidly dephosphorylated in the cytoplasm and effluxes from the cells upon addition of glucose and that glucose inhibits further uptake of TMG. We have developed a vesicular system to analyze this regulatory mechanism and have used electroporation to shock proteins and membrane-impermeable metabolites into the vesicles. Uptake of TMG was dependent on an energy source, effectively provided by intravesicular phosphoenolpyruvate at low concentrations or extravesicular phosphoenolpyruvate at high concentrations. TMG uptake into osmotically shocked vesicles was only weakly inhibited, and expulsion of preaccumulated TMG was only slightly stimulated upon addition of glucose. Intravesicular (but not extravesicular) wild-type HPr of Bacillus subtilis completely restored the regulatory behavior observed in vivo when glucose was present in the external medium. Glucose could be replaced by intravesicular (but not extravesicular) fructose 1,6-diphosphate, gluconate 6-phosphate, or 2-phosphoglycerate, but not by other phosphorylated metabolites, in agreement with the allosteric activating effects of these compounds on HPr(Ser) kinase measured in vitro. Intravesicular mutant HPr(S46A) protein could not promote regulation of lactose permease activity when electroporated into the vesicles regardless of the presence or absence of glucose or the various phosphorylated metabolites, but the HPr(S46D) mutant protein promoted regulation, even in the absence of glucose or a metabolite, and HPr(H15A) was more effective than the wild-type protein in promoting regulation. Intravesicular wild-type and H15A HPrs, but not the S46A or S46D mutant proteins, were found to be phosphorylated by ATP under the conditions which promoted TMG efflux. In toluenized vesicles, the conditions which promoted TMG efflux also promoted TMG-P hydrolysis. These results establish for the first time that HPr serine phosphorylation by the ATP-dependent metabolite-activated HPr kinase regulates the expulsion of intracellular sugar-phosphate as well as the uptake of sugar via the PTS in L. lactis.

Adenosine Triphosphate↗

PubMed1993

[Reaction mechanism of macrolide 2'-phosphotransferase from Escherichia coli to the 2'-modified macrolide antibiotics].

Reaction mechanism of macrolide 2'-phosphotransferase [MPH(2')] from Escherichia coli to the 2'-modified macrolide antibiotics was analyzed by using microbioassay, nuclear magnetic resonance (NMR) spectrometric assay and mass spectrometry. It was found by microbioassay that the 2'-modified macrolide antibiotics as triacetyloleandomycin (TAO), erythromycin ethyl succinate (EME) and erythromycin estolate were inactivated with adenosine triphosphate (ATP) by MPH(2'). The NMR spectrometric assay for the analysis of the reaction with the 2'-modified macrolide antibiotics and MPH(2') was established using guanosine triphosphate, which was higher reaction rate than ATP, as a cofactor. It was clearly shown by NMR spectrometric assay and mass spectrometry that the C2'-side chain of TAO and EME was naturally released in phosphate buffer solution, and then, the C2' position was phosphorylated with GTP by MPH(2').

Anti-Bacterial Agents↗

PubMed1993

Steady state kinetics of mannitol phosphorylation catalyzed by enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system.

The kinetics of mannitol phosphorylation catalyzed by enzyme IImtl of the bacterial P-enolpyruvate-dependent phosphotransferase system are described for three different physical conditions of the enzyme, (i) embedded in the membrane of inside-out (ISO) oriented vesicles, (ii) solubilized and assayed above the critical micellular concentration (cmc) of the detergent, and (iii) solubilized and assayed below the cmc of the detergent. The kinetic characteristics of enzyme IImtl, after solubilization of cytoplasmic membranes or after purification from these membranes are comparable. The mannitol-dependent kinetics at saturating concentration of P-HPr were biphasic both for the solubilized enzyme assayed above the cmc and for the enzyme in ISO vesicles. In contrast, the mannitol-dependent kinetics was monophasic for the solubilized enzyme assayed below the cmc. In the latter case, the maximal rate was about twice as high as observed with the two other conditions. The contribution of the high affinity phase to the maximal rate is lower for enzyme IImtl in ISO vesicles than for the solubilized enzyme. At limiting concentrations of P-HPr, the kinetics is not according to the expected "ping-pong" mechanism.

Cell Membrane↗

PubMed1993

The involvement of the arginine 17 residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site His-15 that is phosphorylated to form N delta 1-P-histidine. The nearby conserved residue, Arg-17, has been replaced by: lysine, histidine, glutamate, glycine, serine, and cysteine. All mutations resulted in impairment of the phosphoacceptor function of HPr with enzyme I: kcat/Km values between 6% (Ser-17) and 0.1% (Glu-17), relative to wild type. Several sugar-specific enzymes II had different responses. Both the Vmax and Km of enzyme IIN-acetylglucosamine were altered, while for enzyme IImannose only Km was affected, except for R17E. For both enzymes, kcat/Km values were between 0.5 and 3%, with R17E being 10-fold lower. Except for R17E, minimal effects were observed for enzyme IImannitol. These results suggest that there are different rate-limiting steps in the enzymes II. Phosphohydrolysis properties and the pKa values for His-15 and phosphorylated His-15 determined by NMR for both wild type and mutant HPrs suggest that Arg-17 is partly responsible for the instability of P-His-15 and the depressed pKa values in wild type HPr. Other feature(s) of the tertiary structure influence the protonation of His-15 and the phosphohydrolysis properties of phosphorylated His-15.

Amino Acid Sequence↗

PubMed1996

[Resistance of cytomegalovirus to ganciclovir: rapid detection of the mutations 460 of the UL97 phosphotransferase].

The substitution of methionine by either isoleucine or valine at residue 460 in the UL97 phosphotransferase has been shown to be responsible for resistance to ganciclovir (GCV) in 30% of resistant cytomegalovirus (CMV) isolates [4]. These substitutions require one nucleotide change in the gene (G- > T 1 380 and A- > G 1378 respectively). The aim of this study was to develop a discriminative PCR assay for rapid detection of these DNA changes. A PCR assay was duplicated in parallel for each mutation; to detect G- > T 1380 each reaction mixture contained primer VSUL14 and either primer LNW to distinguish wild type residues or LNM to distinguish mutant residues, and for A- > G 1378 primers were VSUL8 and either MCMW to detect wild type sequences or MCMM to detect mutated residues. For optimal discrimination, primers MCMW and MCMM were designed with a mismatch at position 3'-1. The reference strains AD169, Davis and Towne, a laboratory GCV-resistant mutant RCL1.7, and 33 CMV isolates (10 resistant, 2 indetermined and 21 sensitive) were tested by PCR. AD169, Davis and Towne, and 30 isolates were amplified only with non modified primers, and the absence of 460 mutations was confirmed by sequencing. Two isolates P1 and P2, from a transplanted patient were amplified with both MCMM and MCMW: sequencing analysis shown the presence of a mixture of strain, one of them harbouring A- > G 1378 mutation. One resistant strain was amplified neither with MCMM nor with MCMW: a C- > T silent mutation at nt 1368 was present. As sequencing analysis confirmed PCR results, discriminative PCR enables isolates to be rapidly assessed for the presence or absence of 460 mutations. Moreover, it can distinguish Met to Val from Met to Ile mutations, and allows the analysis of mixtures of sensitive ad resistant strains.

AIDS-Related Opportunistic Infections↗

PubMed1996

Aggregation of banana pyrophosphate fructose 6-phosphate 1-phosphotransferase by glycerol.

Addition of glycerol during purification of banana (Musaceae, Musa cavendishii) pyrophosphate fructose 6-phosphate 1-phosphotransferase [(PFP), EC 2.7.1.90] initiated molecular aggregation of the enzyme. The aggregation process was dependent on the glycerol concentration. The native enzyme (66 kDa molecular mass) showed enhanced activity at 3% (V/V) or less of glycerol concentration. Glycerol concentration between 4 and 5% (V/V) affected a gradual and sequential aggregation of native form of the enzyme. These aggregated forms had molecular masses of 135, 200 and 270 kDa. The 135 and 200 kDa forms were stable for about 72 hrs and prolonged storage over 2 weeks resulted in the formation of the 270 kDa form. Concentration over 5% could reduce the time required for aggregation. Fru2.6 bis P activated the enzyme over ten fold, but did not help in the aggregation process. Studies on the role of glycerol on PFP specific activity suggested a difference in the activation process compared to that by Fru2.6bis P. Replacement of Hepes buffer by Tris increased the Fru2.6 bis P requirement for maximum activation by around 10 fold. Removal of glycerol from the buffer media resulted in almost complete inactivation of the enzyme.

Cellulose↗

PubMed1998

Expression of the membrane protein glycophorin A as a fusion with the antibiotic resistance protein neomycin phosphotransferase II.

The gene for the integral membrane protein glycophorin A (GPA) was cloned in frame to the 5' end of the antibiotic resistance gene, neomycin phosphotransferase II (NPT). Protein expression was achieved in Escherichia coli as well as in mammalian cells. In case of Chinese hamster ovary cells (CHO) the resistant populations were analyzed 2 weeks after transfection; the amount of GPA-NPT fusion protein produced was constant from experiment to experiment. Neomycin resistance was directly correlated with GPA expression, thus allowing the direct selection for a stable GPA-expressing cell population without the need of a cloning step. The amount of GPA-NPT produced was further increased by weakening the specific NPT enzymatic activity via site-directed mutagenesis. Detection was simplified by the fact that all different fusion proteins could be detected by the same anti-NPT antibody. This approach may be also applicable to other membrane proteins.

Animals↗

PubMed1983

The mechanism of inducer exclusion. Direct interaction between purified III of the phosphoenolpyruvate:sugar phosphotransferase system and the lactose carrier of Escherichia coli.

A hypothesis for the regulation of some sugar transport systems by the bacterial phosphoenolpyruvate:sugar transport system postulates an interaction between III of this system and the carrier whose activity is regulated. We have studied this interaction in more detail, employing one of these transport systems, the lactose carrier of Escherichia coli. Purified III of the phosphotransferase system interacted directly with the lactose carrier. The binding of III to lactose carrier required the presence of the non-phosphorylated form of III and substrates of the carrier and exhibited a stoichiometry of 1.2+/- 0.2 mol III/mol lactose carrier. The K(d) of lactose carrier for III was 10 +/- 5 microM. III is apparently unable to interact with a mutant lactose carrier which still binds but does not transport galactosides. The binding of III to the lactose carrier results in a 3.5-fold increase in the apparent affinity of galactosides for the carrier. Significantly, the binding of III to the lactose carrier results in an inhibition of galactoside translocation both in membrane vesicles and liposomes reconstituted with the purified lactose carrier. This inhibition may thus be the basis for the well-documented phenomenon of inducer exclusion.

Journal Article↗

PubMed1998

Isolation and characterization of mutated mitochondrial GTP:AMP phosphotransferase.

GTP:AMP phosphotransferase (adenylate kinase isozyme 3, AK3) mutants were obtained by using the ability of AK3 to complement a temperature-sensitive mutation of Escherichia coli adenylate kinase (AKe). Five mutants, P16L, G19S, G91D, G91S, and P93L, had mutation sites located at two loops that are involved in substrate binding of the enzyme. P16L and G19S bearing changes at the first loop showed reduced affinity for both GTP and AMP, the extent of reduction being slightly higher for GTP than AMP. In contrast, G91S and P93L having alterations at the second loop had lower affinities for AMP. Only the alterations at the second loop strongly influenced the Vmax value of the enzyme. Another mutant, D163N, had a substitution at the site forming a salt bridge in adenylate kinase isozyme 1 (AK1), which influenced the Vmax as well as the Km values for both substrates. The kinetic characteristics of these mutants were comparable to those of the corresponding AK1 or AKe mutants. Furthermore, from the results of mutations T201P and T201A that had alterations in all the kinetic parameters of AK3 and from a comparison with the structure and the kinetic parameters of AKe, we expect that a residue(s) around Thr201 is involved in recognition of the base of nucleoside triphosphate.

Adenylate Kinase↗

PubMed1979

Nucleoside phosphotransferase of chick embryo.

This paper describes a purification procedure and some properties of a nonspecific nucleoside phosphotransferase of chick embryo, an activity which catalyzes the transfer of chick embryo, an activity which catalyzes the transfer of the phosphate ester from a deoxyribonucleotide or a pyrimidine ribonucleotide to a deoxyribonucleoside acceptor. The enzyme is very unstable to heat, dilution and dialysis and it is almost entirely inactivated by DEAE-cellulose chromatography or gel filtration. A marked enhancement in its stability is caused by numerous nucleotides. In these experiments at least 920-fold purification was obtained by using dTTP (50 microM) as nucleotide protector. The enzyme, purified in presence of dTTP, has a molecular weight about 270,000, an isoelectric point of 6.27, a pH optimum of 8.8 and is stable at 37 degrees C at least for 10 min. In absence of nucleotide protector, nucleoside phosphofranserferase is connected at 37 degrees C or by gel filtration in a very small active form with a lower molecular weight (about 30,000) and a pH optimum of 7.6.

Animals↗

PubMed2006

Abnormal expressions of the subunits of the UDP-N-acetylglucosamine: lysosomal enzyme, N-acetylglucosamine-1-phosphotransferase, result in the formation of cytoplasmic vacuoles resembling those of the I-cells.

Defects in the multimeric enzyme, UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GNPT), result in the diseases of mucolipidosis (ML). This enzyme generates the mannose 6-phosphate residues on newly synthesized lysosomal enzymes for the efficient receptor-mediated transport to lysosomes. The enzyme contains alpha/beta and gamma subunits. Mutations in the alpha/beta subunit result in the classical ML II and IIIA, while defects in the gamma subunit results in the clinically milder ML IIIC. I-cells, a distinct histological feature characterized by the presence of abnormal cytoplasmic vacuoles, are detected in many cell types, most noticeably, in ML II patients. In this study, we investigated the interactions of the alpha/beta and gamma subunits in the pathogenesis of I-cells. We noted low and deranged alpha/beta subunit expressions in human mucolipidosis cell lines. Unexpectedly, high gamma subunit expressions were also observed. In normal mouse fibroblasts, when alpha/beta subunit was suppressed, abnormal cytoplasmic vacuoles were induced, and up-regulation of the gamma subunit was also observed. On the other hand, suppressing the gamma subunit resulted in biphasic responses of the alpha/beta subunit, while abnormal cytoplasmic vacuoles were not formed, regardless of the expression levels of the alpha/beta subunit. Our data suggest reciprocal feedback mechanisms between alpha/beta and the gamma subunits. A fine balance of the expressions of these subunits may play an important role in the formation of I-cells in this group of lysosomal storage disorders.

Cell Culture Techniques↗