Characterization of the P450 monooxygenase NysL, responsible for C-10 hydroxylation during biosynthesis of the polyene macrolide antibiotic nystatin in Streptomyces noursei

The nysL gene, encoding a putative P450 monooxygenase, was identified in the nystatin biosynthetic gene cluster of Streptomyces noursei. Although it has been proposed that NysL is responsible for hydroxylation of the nystatin precursor, experimental evidence for this activity was lacking. The nysL gene was inactivated in S. noursei by gene replacement, and the resulting mutant was shown to produce 10-deoxynystatin. Purification and an in vitro activity assay for 10-deoxynystatin demonstrated its antifungal activity being equal to that of nystatin. The NysL protein was expressed heterologously in Escherichia coli as a His-tagged protein and used in an enzyme assay with 10-deoxynystatin as a substrate. The results obtained clearly demonstrated that NysL is a hydroxylase responsible for the post-polyketide synthase modification of 10-deoxynystatin at position C-10. Kinetic studies with the purified recombinant enzyme allowed determination of K(m) and k(cat) and revealed no inhibition of recombinant NysL by either the substrate or the product. These studies open the possibility for in vitro evolution of NysL aimed at changing its specificity, thereby providing new opportunities for engineered biosynthesis of novel nystatin analogues hydroxylated at alternative positions of the macrolactone ring.     

Analysis of the Mycosamine Biosynthesis and Attachment Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455

The polyene macrolide antibiotic nystatin produced by Streptomyces noursei contains a deoxyaminosugar mycosamine moiety attached to the C-19 carbon of the macrolactone ring through the β-glycosidic bond. The nystatin biosynthetic gene cluster contains three genes, nysDI, nysDII, and nysDIII, encoding enzymes with presumed roles in mycosamine biosynthesis and attachment as glycosyltransferase, aminotransferase, and GDP-mannose dehydratase, respectively. In the present study, the functions of these three genes were analyzed. The recombinant NysDIII protein was expressed in Escherichia coli and purified, and its in vitro GDP-mannose dehydratase activity was demonstrated. The nysDI and nysDII genes were inactivated individually in S. noursei, and analyses of the resulting mutants showed that both genes produced nystatinolide and 10-deoxynystatinolide as major products. Expression of the nysDI and nysDII genes in trans in the respective mutants partially restored nystatin biosynthesis in both cases, supporting the predicted roles of these two genes in mycosamine biosynthesis and attachment. Both antifungal and hemolytic activities of the purified nystatinolides were shown to be strongly reduced compared to those of nystatin, confirming the importance of the mycosamine moiety for the biological activity of nystatin.     

Carboxyl-terminal domain characterization of polyene-specific P450 hydroxylase in <Emphasis Type="Italic">Pseudonocardia autotrophica</Emphasis>

A polyene compound NPP identified in Pseudonocardia autotrophica was shown to contain an aglycone identical to nystatin, but to harbor a unique disaccharide moiety that led to higher solubility and reduced hemolytic activity. Recently, it was revealed that the final step of NPP (nystatin-like polyene) biosynthesis is C10 regio-specific hydroxylation by the cytochrome P450 hydroxylase (CYP) NppL (Kim et al. [7]). Through mutation and cross-complementation, here we found that NppL preferred a polyene substrate containing a disaccharide moiety for C10 hydroxylation, while its orthologue NysL involved in nystatin biosynthesis showed no substrate preference toward mono- and disaccharide moieties, suggesting that two homologous polyene CYPs, NppL and NysL might possess a unique domain recognizing a sugar moiety. Two hybrid NppL constructs containing the C-terminal domain of NysL exhibited no substrate preference toward 10-deoxy NPP and 10-deoxy nystatin-like NysL, implying that the C-terminal domain plays a major role in differentiating the sugar moiety responsible for substrate specificity. Further C-terminal domain dissection of NppL revealed that the last fifty amino acids play a critical role in determining substrate specificity of polyene-specific hydroxylation, setting the stage for the biotechnological application of hydroxyl diversification for novel polyene biosynthesis in actinomycetes.     

Effect of glucose limitation and specific mutations in the module 5 enoyl reductase domains in the nystatin and amphotericin polyketide synthases on polyene macrolide biosynthesis

Enoyl reductase (ER) domains in module 5 of nystatin and amphotericin polyketide synthase (PKS) are responsible for reduction of the C28–C29 unsaturated bond on the nascent polyketide chain during biosynthesis of both macrolides, resulting in production of tetraenes nystatin A₁ and amphotericin A, respectively. Data obtained in fermentations under glucose limitation conditions demonstrated that the efficiency of the ER5 domain can be influenced by carbon source availability in the amphotericin producer Streptomyces nodosus, but not in the nystatin producer Streptomyces noursei. Two S. noursei ER5 domain mutants were constructed, GG5073SP and S5016N, both producing the heptaene nystatin analogue S44HP with unsaturated C28–C29 bond. While the GG5073SP mutant, with altered ER5 NADPH binding site, produced S44HP exclusively, the S5016N mutant synthesized a mixture of nystatin and S44HP. Comparative studies on the S5016N S. noursei mutant and S. nodosus, both producing mixtures of tetraenes and heptaenes, revealed that the ratio between these two types of metabolites was significantly more affected by glucose limitation in S. nodosus. These data suggest that mutation S5016N in NysC “locks” the ER5 domain in a state of intermediate activity which, in contrast to the ER5 domain in the amphotericin PKS, is not significantly influenced by physiological conditions.     

Biosynthesis of the polyene macrolide antibiotic nystatin in Streptomyces noursei

The polyene macrolide antibiotic nystatin, produced commercially by the bacterium Streptomyces noursei, is an important antifungal agent used in human therapy for treatment of certain types of mycoses. Early studies on nystatin biosynthesis in S. noursei provided important information regarding the precursors utilised in nystatin biosynthesis and factors affecting antibiotic yield. New insights into the enzymology of nystatin synthesis became available after the gene cluster governing nystatin biosynthesis in S. noursei was cloned and analysed. Six large polyketide synthase proteins were implicated in the formation of the nystatin macrolactone ring, while other enzymes, such as P450 monooxygenases and glycosyltransferase, were assumed responsible for ring decoration. The latter data, supported by analysis of the polyene mixture synthesised by the nystatin producer, helped elucidate the complete nystatin biosynthetic pathway. This information has proved useful for engineered biosynthesis of novel nystatin analogues, suggesting a plausible route for the generation of potentially safer and more efficient antifungal drugs.     

An improved assay method for a pseudomurein-degrading enzyme of Methanobacterium wolfei and the Protoplast formation of Methanobacterium thermoautotrophicum by the enzyme

An improved assay method of a pseudomurein-degrading enzyme and its properties are described. The pseudomurein-degrading enzyme purified from Methanobacterium wolfei autolysate under an anoxic condition was assayed with the cell wall of Methanobacterium thermoautotrophicum as a substrate. By this improved method the enzyme activity was measured quantitatively and reproducibly. Moreover, the cell wall substrate can be stored in a freezer and used as needed, and the time required for an assay was as short as 1 h. The optimum pH and temperature of the enzyme was pH 6.8-7.4 and 75°C, respectively. Although the enzyme lost 50% of the activity upon heating at 75°C for 10 min in the absence of the cell wall substrate, it was more stable against heat inactivation in the presence of the substrate. Furthermore the inactivated enzyme recovered some of the activity by incubating with the substrate. Although the enzyme lost most of the activity under aerobic conditions, the activity was recovered under reducing conditions with Na₂S·9H₂O or DTT (dithiothreitol). The enzyme was also purified under aerobic conditions retaining the same specific activity as the anoxically purified enzyme. Using the partially purified enzyme the conditions preparing protoplasts of M. thermoautotrophicum was established.     

Heterologous extracellular expression and initial characterization of the peroxisomal catalase from the methylotrophic yeast Hansenula polymorpha in Pichia pastoris

Catalase is well known to eliminate H₂O₂ in cells and reduces the toxicity of peroxide compounds. A catalase gene HpCAT1 of methylotrophic yeast Hansenula polymorpha without the part coding the native signal peptide was cloned into expression vector pYM3165 and then integrated into genome of Pichia pastoris GS115 by electroporation. The result of the enzyme activity assay and SDS-PAGE demonstrated that the recombinant protein (HpCAT1) of H. polymorpha was extracellularly expressed in P. pastoris. The expressed catalase was recovered from the culture supernatant of P. pastoris GS115 and purified by (NH₄)₂SO₄ fractionation and Ni-NTA affinity chromatography. The main biochemical properties of the recombinant protein HpCAT1, such as thermodependence and thermostability, pH optimum and pH stability, as well as the effect of metal ions and chemicals, were characterized. With H₂O₂ as the substrate, HpCAT1 displayed pH and temperature optima of ～2.6 and 45°C, respectively. The recombinant HpCAT1 activity was inhibited by 1 mM Hg²⁺ and Cu²⁺, but was highly enhanced by 1.0 mM Fe²⁺.     

Cloning and characterization of archaeal type I signal peptidase from Methanococcus voltae

Archaeal protein trafficking is a poorly characterized process. While putative type I signal peptidase genes have been identified in sequenced genomes for many archaea, no biochemical data have been presented to confirm that the gene product possesses signal peptidase activity. In this study, the putative type I signal peptidase gene in Methanococcus voltae was cloned and overexpressed in Escherichia coli, the membranes of which were used as the enzyme source in an in vitro peptidase assay. A truncated, His-tagged form of the M. voltae S-layer protein was generated for use as the substrate to monitor the signal peptidase activity. With M. voltae membranes as the enzyme source, signal peptidase activity in vitro was optimal between 30 and 40°C; it was dependent on a low concentration of KCl or NaCl but was effective over a broad concentration range up to 1 M. Processing of the M. voltae S-layer protein at the predicted cleavage site (confirmed by N-terminal sequencing) was demonstrated with the overexpressed archaeal gene product. Although E. coli signal peptidase was able to correctly process the signal peptide during overexpression of the M. voltae S-layer protein in vivo, the contribution of the E. coli signal peptidase to cleavage of the substrate in the in vitro assay was minimal since E. coli membranes alone did not show significant activity towards the S-layer substrate in in vitro assays. In addition, when the peptidase assays were performed in 1 M NaCl (a previously reported inhibitory condition for E. coli signal peptidase I), efficient processing of the substrate was observed only when the E. coli membranes contained overexpressed M. voltae signal peptidase. This is the first proof of expressed type I signal peptidase activity from a specific archaeal gene product.     

Effect of temperature and sorbitol in improving the solubility of carboxylesterases protein CpCE-1 from Cydia pomonella and biochemical characterization

Carboxylesterases (CEs) are enzymes responsible for the detoxification of insecticides in insects. In the Cydia pomonella, CEs are involved in synthetic pyrethroid, neonicotinoid, carbamate, and organophosphate detoxification. However, functional overexpression of CEs proteins in Escherichia coli systems often results in insoluble proteins. In this study, we expressed the fusion protein CpCE-1 in E. coli BL21 (DE3). This recombinant protein was overexpressed as inclusion bodies at 37 °C whereas it produced a higher percentage of soluble protein at lower growth temperatures. Production of soluble proteins and enzyme activity increased in the presence of sorbitol in the growth medium. The fusion protein was purified from the lysate supernatant using a Ni²⁺-NTA agarose gel column. The enzyme exhibited a higher affinity and substrate specificity for α-naphthyl acetate (α-NA), with k _cat/K _m of 100 s^?1 μM^?1 for α-NA, and the value is 29.78 s^?1 μM^?1 for β-naphthyl acetate. The V _max and K _m were also determined to be 12.9 μmol/min/mg protein and 13.4 μM using substrate α-NA. The optimum pH was 7.0 and temperature was 25 °C. An enzyme inhibition assay shows that PMSF and DEPC strongly inhibit the enzyme activity, while the metal ions Cu²⁺ and Mg²⁺ significantly activated the activity. More importantly, cypermethrin, methomyl, and acephate were found to suppress enzyme activity. The data demonstrated here provide information for heterologous expression of soluble protein and further study on insecticide metabolism in C. pomonella in vitro. This is the first report of the characterization of CEs protein from C. pomonella.     

In Vivo Characterization of Dimethylsulfoniopropionate Lyase in the Fungus Fusarium lateritium

A fungus, Fusarium lateritium, with dimethylsulfoniopropionate (DMSP) lyase activity was isolated from both seawater and a salt marsh due to its ability to grow on DMSP (with the evolution of dimethyl sulfide) as the sole source of carbon. This is the first reported case of DMSP lyase activity in a fungus. Several other common fungal genera tested did not have DMSP lyase activity. DMSP was taken up more rapidly by F. lateritium than it was utilized, leading to its intracellular accumulation. Inhibitor studies with nystatin and cyanide indicated that DMSP uptake was an energy-dependent process. The lyase was inducible by its substrate, DMSP (K_m, 1.2 mM), and by the substrate analogs choline and glycine betaine. During induction, DMSP lyase activity increased with time and then dropped rapidly. This loss of activity could be prevented by spiking the culture with fresh DMSP or choline. The V_max for DMSP lyase was 34.7 mU · mg of protein⁻¹. The inhibitory effects of nystatin, and p-chloromercuriphenylsulfonate on DMSP lyase activity suggested that the enzyme is cytosolic. Because plants like Spartina (a marsh grass) and marine algae contain high concentrations of DMSP, we speculate that DMSP-utilizing fungi may be involved in their decay.     

FK506 Binding Protein from the Hyperthermophilic Archaeon Pyrococcus horikoshii Suppresses the Aggregation of Proteins in Escherichia coli

The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (k_cat/K_m) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15°C. Besides this low PPIase activity, PhFKBP29 showed chaperone-like protein folding activity which enhanced the refolding yield of chemically unfolded rhodanese in vitro. In addition, it suppressed thermal protein aggregation in a temperature range of 45 to 100°C. When the PhFKBP29 gene was coexpressed with the recombinant Fab fragment gene of the anti-hen egg lysozyme antibody in the cytoplasm of E. coli, whose expressed product tended to form an inactive aggregate in E. coli, it improved the yield of the soluble Fab fragments with antibody specificity. PhFKBP29 exerted protein folding and aggregation suppression in E. coli cells.     

New Nystatin-Related Antifungal Polyene Macrolides with Altered Polyol Region Generated via Biosynthetic Engineering of Streptomyces noursei

Polyene macrolide antibiotics, including nystatin and amphotericin B, possess fungicidal activity and are being used as antifungal agents to treat both superficial and invasive fungal infections. Due to their toxicity, however, their clinical applications are relatively limited, and new-generation polyene macrolides with an improved therapeutic index are highly desirable. We subjected the polyol region of the heptaene nystatin analogue S44HP to biosynthetic engineering designed to remove and introduce hydroxyl groups in the C-9-C-10 region. This modification strategy involved inactivation of the P450 monooxygenase NysL and the dehydratase domain in module 15 (DH15) of the nystatin polyketide synthase. Subsequently, these modifications were combined with replacement of the exocyclic C-16 carboxyl with the methyl group through inactivation of the P450 monooxygenase NysN. Four new polyene macrolides with up to three chemical modifications were generated, produced at relatively high yields (up to 0.51 g/liter), purified, structurally characterized, and subjected to in vitro assays for antifungal and hemolytic activities. Introduction of a C-9 hydroxyl by DH15 inactivation also blocked NysL-catalyzed C-10 hydroxylation, and these modifications caused a drastic decrease in both antifungal and hemolytic activities of the resulting analogues. In contrast, single removal of the C-10 hydroxyl group by NysL inactivation had only a marginal effect on these activities. Results from the extended antifungal assays strongly suggested that the 9-hydroxy-10-deoxy S44HP analogues became fungistatic rather than fungicidal antibiotics.     

HPLC method for the determination of phytochelatin synthase activity specific for soft metal ion chelators

Phytochelatins (PCs) are nonprotein peptides with the general structure (γ-Glu-Cys)_n-Gly (PC_n), where n is greater than or equal to 2. They are synthesized through a reaction catalyzed by phytochelatin synthase (PCS) in the presence of metal cations and using the tripeptide glutathione (γ-Glu-Cys-Gly) and/or previously synthesized PC_n as the substrate. Here, a highly sensitive assay for PCS activity was devised, in which the dequenching of Cu(I)-bathocuproinedisulfonate complexes was used in the detection system of a reversed-phase high-performance liquid chromatograph. Using recombinant PCS from the higher plant Arabidopsis thaliana (rAtPCS1), this assay system was capable of determining PCS activity based on an amount of the enzyme preparation that was 100-fold less than that required for the 5,5′-dithiobis(2-nitrobenzoic acid) assay method. Although adsorption of the enzyme onto the reaction vessel hindered accurate activity determination, the inclusion of bovine serum albumin successfully resolved this issue. This method is a powerful tool for investigating PCS enzyme mechanisms with respect to the roles of metal ions.     

A long-wavelength fluorescent substrate for continuous fluorometric determination of α-mannosidase activity: Resorufin α-d-mannopyranoside

A simple and reliable continuous assay for measurement of α-mannosidase activity is described and demonstrated for analysis with two recombinant human enzymes using the new substrate resorufin α-d-mannopyranoside (Res-Man). The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585 nm with excitation at 571 nm and has a pK_a of 5.8, allowing continuous measurement of fluorescence turnover at or near physiological pH values for human lysosomal and Drosophila Golgi α-mannosidases. The assay performed using recombinant Drosophila Golgi α-mannosidase (dGMII) has been shown to give the kinetic parameters K_m of 200 μM and V_max of 11 nmol/min per nmol dGMII. Methods for performing the assay using several concentrations of the known α-mannosidase inhibitor swainsonine are also presented, demonstrating a potential for use of the assay as a simple method for high-throughput screening of inhibitors potentially useful in cancer treatment.     

Identification of a New 1,4-beta-D-xylosidase <Emphasis Type="Italic">Pae</Emphasis>1263 from the Whole Genome Sequence of <Emphasis Type="Italic">Paenibacillus terrae</Emphasis> HPL-003

The gene of Pae1263 (2,196 bp, 732 aa) was found from the full-length sequence analysis of bacterium Paenibacillus terrae HPL-003 isolated from soil on Gara Mountain in Korea (CP003107, our previous study). Among the 20 open reading frames (ORFs) related with the xylose substrate, only the recombinant enzyme of ORF Pae1263 showed a 1,4-beta-D-xylosidase activity when all of the ORFs were transformed into E. coli. This gene is considered to be a new 1,4-beta-D-xylosidase because it has up to 93% similarity with other genes of ZP_10240221.1 from Lactococcus raffinolactis 4877 and ZP_11237858.1 from Paenibacillus peoriae in the GenBank blast search. The enzyme activity was confirmed by HPLC in which xylose was produced from xylobiose as a substrate by this recombinant enzyme. Mass production of the recombinant enzyme was done with the construction of the pET22(+)- Pae1263-6H expression vector system from E. coli. This new 1,4-beta-D-xylosidase was highly active at 50°C in a pH range between 6.0 and 8.0 and had thermo-stability for at least 24 h at 50°C and a K _m and V _max of 6.42 mg/mL and 75.76 U/mg on a xylobiose substrate, respectively.     

A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification,characterization, and expression

The carboxylesterase, a 34 kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 85 °C and 8.0, respectively. The enzyme showed remarkable thermostability: 41% of its activity remained after 5 days of incubation at 80 °C. In addition, the purified enzyme exhibited stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity towards various PNP esters and short acyl chain triacylglycerols such as tributyrin (C_4:0). Among the PNP esters tested, the best substrate was PNP-caprylate (C₈) with K_m and k_cat values of 71 μM and 14,700 s⁻¹, respectively. The carboxylesterase gene consisted of 915 bp corresponding to 305 amino acid residues. We demonstrated that active recombinant S. solfataricus carboxylesterase could be expressed in Escherichia coli. The enzyme was identified as a serine esterase belonging to mammalian hormone-sensitive lipases (HSL) family and contained a catalytic triad composed of serine, histidine, and aspartic acid in the active site.     

Application of an airlift bioreactor to the nystatin biosynthesis

Pilot plant studies were performed using a concentric-tube airlift bioreactor of 2.5 m³ fermentation volume. The results have proven the relative merits of such a system in the biosynthesis of nystatin, produced by Streptomyces noursei, in submerged aerobic cultivation and batch operation mode. The results were compared to those obtained in a pilot-scale stirred tank bioreactor of 3.5 m³ fermentation volume. The fermentation processes in the two fermentation devices were similar with respect to substrate utilization, biomass production and nystatin biosynthesis. In the riser section, the dissolved oxygen concentration was higher than that in the downcomer. The volumetric oxygen mass transfer coefficient was dependent on the rheological behaviour of the biosynthesis liquids, which was not constant during the fermentation process. The total energy consumption for nystatin production in the airlift bioreactor was 56% of that in the stirred tank, while the operating costs represented 78% of those in the stirred tank bioreactor.     

Characterization of Native and Recombinant Forms of an Unusual Cobalt-Dependent Proline Dipeptidase (Prolidase) from the Hyperthermophilic Archaeon Pyrococcus furiosus

Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co²⁺ ions (K_d, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co²⁺ could be replaced by Mn²⁺ (resulting in a 25% decrease in activity) but not by Mg²⁺, Ca²⁺, Fe²⁺, Zn²⁺, Cu²⁺, or Ni²⁺. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100°C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P. furiosus. Furthermore, the K_m values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater V_max value than the native protein. The amino acid sequence of P. furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.     

Thioesterase activity and subcellular localization of acylprotein thioesterase 1/lysophospholipase 1

Acylprotein thioesterase 1 (APT1), also known as lysophospholipase 1, is an important enzyme responsible for depalmitoylation of palmitoyl proteins. To clarify the substrate selectivity and the intracellular function of APT1, we performed kinetic analyses and competition assays using a recombinant human APT1 (hAPT1) and investigated the subcellular localization. For this purpose, an assay for thioesterase activity against a synthetic palmitoyl peptide using liquid chromatography/mass spectrometry was established. The thioesterase activity of hAPT1 was most active at neutral pH, and did not require Ca²⁺ for its maximum activity. The K_M values for thioesterase and lysophospholipase (against lysophosphatidylcholine) activities were 3.49 and 27.3 μM, and the V_max values were 27.3 and 1.62 μmol/min/mg, respectively. Thus, hAPT1 revealed much higher thioesterase activity than lysophospholipase activity. One activity was competitively inhibited by another substrate in the presence of both substrates. Immunocytochemical and Western blot analyses revealed that endogenous and overexpressed hAPT1 were mainly localized in the cytosol, while some signals were detected in the plasma membrane, the nuclear membrane and ER in HEK293 cells. These results suggest that eliminating palmitoylated proteins and lysophospholipids from cytosol is one of the functions of hAPT1.