Distinct acyl protein transferases and thioesterases control surface expression of calcium-activated potassium channels

Protein palmitoylation is rapidly emerging as an important determinant in the regulation of ion channels, including large conductance calcium-activated potassium (BK) channels. However, the enzymes that control channel palmitoylation are largely unknown. Indeed, although palmitoylation is the only reversible lipid modification of proteins, acyl thioesterases that control ion channel depalmitoylation have not been identified. Here, we demonstrate that palmitoylation of the intracellular S0-S1 loop of BK channels is controlled by two of the 23 mammalian palmitoyl-transferases, zDHHC22 and zDHHC23. Palmitoylation by these acyl transferases is essential for efficient cell surface expression of BK channels. In contrast, depalmitoylation is controlled by the cytosolic thioesterase APT1 (LYPLA1), but not APT2 (LYPLA2). In addition, we identify a splice variant of LYPLAL1, a homolog with ～30% identity to APT1, that also controls BK channel depalmitoylation. Thus, both palmitoyl acyltransferases and acyl thioesterases display discrete substrate specificity for BK channels. Because depalmitoylated BK channels are retarded in the trans-Golgi network, reversible protein palmitoylation provides a critical checkpoint to regulate exit from the trans-Golgi network and thus control BK channel cell surface expression.     

Dynamic Palmitoylation Links Cytosol-Membrane Shuttling of Acyl-protein Thioesterase-1 and Acyl-protein Thioesterase-2 with That of Proto-oncogene H-Ras Product and Growth-associated Protein-43

Acyl-protein thioesterase-1 (APT1) and APT2 are cytosolic enzymes that catalyze depalmitoylation of membrane-anchored, palmitoylated H-Ras and growth-associated protein-43 (GAP-43), respectively. However, the mechanism(s) of cytosol-membrane shuttling of APT1 and APT2, required for depalmitoylating their substrates H-Ras and GAP-43, respectively, remained largely unknown. Here, we report that both APT1 and APT2 undergo palmitoylation on Cys-2. Moreover, blocking palmitoylation adversely affects membrane localization of both APT1 and APT2 and that of their substrates. We also demonstrate that APT1 not only catalyzes its own depalmitoylation but also that of APT2 promoting dynamic palmitoylation (palmitoylation-depalmitoylation) of both thioesterases. Furthermore, shRNA suppression of APT1 expression or inhibition of its thioesterase activity by palmostatin B markedly increased membrane localization of APT2, and shRNA suppression of APT2 had virtually no effect on membrane localization of APT1. In addition, mutagenesis of the active site Ser residue to Ala (S119A), which renders catalytic inactivation of APT1, also increased its membrane localization. Taken together, our findings provide insight into a novel mechanism by which dynamic palmitoylation links cytosol-membrane trafficking of APT1 and APT2 with that of their substrates, facilitating steady-state membrane localization and function of both.     

Depalmitoylation of endothelial nitric-oxide synthase by acyl-protein thioesterase 1 is potentiated by Ca(2+)-calmodulin.

Protein palmitoylation represents an important mechanism governing the dynamic subcellular localization of many signaling proteins. Palmitoylation of endothelial nitric-oxide synthase (eNOS) promotes its targeting to plasmalemmal caveolae; agonist-promoted depalmitoylation leads to eNOS translocation. Depalmitoylation and translocation of eNOS modulate the agonist response, but the pathways that regulate eNOS palmitoylation and depalmitoylation are poorly understood. We now show that the newly characterized acyl-protein thioesterase 1 (APT1) regulates eNOS depalmitoylation. Immunoblot analyses indicate that APT1 is expressed in bovine aortic endothelial cells, which express eNOS. APT1 overexpression appears to accelerate the depalmitoylation of eNOS in COS-7 cells cotransfected with eNOS and APT1 cDNAs. Additionally, purified recombinant APT1 depalmitoylates eNOS assayed in biological membranes isolated from endothelial cells biosynthetically labeled with [(3)H]palmitate or COS-7 cells transfected with eNOS cDNA. More important, the APT1-catalyzed depalmitoylation of palmitoyl-eNOS is potentiated by Ca(2+)-calmodulin (CaM), a key allosteric activator of eNOS. In contrast, APT1-catalyzed depalmitoylation of the G protein Galpha(s) is unaffected by Ca(2+)-CaM. Furthermore, caveolin, a palmitoylated membrane protein, does not appear to be a substrate for APT1. Taken together, these results support a role for APT1 in the regulation of eNOS depalmitoylation and suggest that Ca(2+)-CaM activation of eNOS renders the enzyme more susceptible to APT1-catalyzed depalmitoylation.     

Protein Depalmitoylation Is Induced by Wnt5a and Promotes Polarized Cell Behavior

Wnt5a signaling regulates polarized cell behavior, but the downstream signaling events that promote cell polarity are not well understood. Our results show that Wnt5a promotes depalmitoylation of the melanoma cell adhesion molecule (MCAM) at cysteine 590. Mutation of Cys-590 to glycine is sufficient to polarize MCAM localization, similar to what is observed with Wnt5a stimulation. Inhibition of the depalmitoylating enzyme APT1 blocks Wnt5a-induced depalmitoylation, asymmetric MCAM localization, and cell invasion. Directly altering expression of the basal protein palmitoylation machinery is sufficient to promote cell invasion. Additionally, cancer mutations in palmitoyltransferases decrease MCAM palmitoylation and have impaired ability to suppress cell invasion. Our results provide evidence that Wnt5a induces protein depalmitoylation, which promotes polarized protein localization and cell invasion.     

Palmitoylation: A new mechanism for control of NCX1 function

A paper by Gök et al., identified Zinc Finger DHHC-Type Palmitoyltransferase 5 (zDHHC5) and Acyl-Protein Transferase 1 (APT1) as the enzymes responsible for the dynamic palmitoylation of NCX1. Palmitoylation occurs at the cell surface and increases the affinity of NCX1 for lipid rafts. Additionally, they discovered that palmitoylation controls the affinity of NCX1 for exchange inhibitory protein (XIP) and regulates intracellular calcium concentration. These findings provide new insights into endogenous control of NCX1 function and will drive future investigations directed at understanding its full potential.     

Topical review: Shedding light on molecular and cellular consequences of NCX1 palmitoylation

Palmitoylation is the post-translational, covalent and reversible conjugation of a 16C saturated fatty acid to cysteine residues of proteins. The sodium calcium exchanger NCX1 is palmitoylated at a single cysteine residue in its large regulatory intracellular loop. Inactivation, mediated by the NCX1 inhibitory region XIP, is drastically impaired in unpalmitoylatable NCX1. The ability of XIP to bind and inactivate NCX1 is largely determined by NCX1 palmitoylation, which induces local conformational changes in the NCX1 intracellular loop to enable XIP to engage its binding site. Consequently, NCX1 palmitoylation regulates intracellular calcium by changing NCX1 sensitivity to inactivation. NCX1 palmitoylation is a dynamic phenomenon which is catalyzed by the palmitoyl acyl transferase zDHHC5 and reversed by the thioesterase APT1, with the switch between palmitoylated and depalmitoylated states, which has profound effects on NCX1 lipid interactions, influenced by NCX1 conformational poise. Herein we review the molecular and cellular consequences of NCX1 palmitoylation and its physiological relevance and highlight the importance of palmitoylation for NCX1 activity. We discuss the cellular control of protein palmitoylation and depalmitoylation, the relationship between lipid microdomains and lipidated and phospholipid binding proteins, and highlight the important unanswered questions in this emerging field.     

Palmitoylation-induced Aggregation of Cysteine-string Protein Mutants That Cause Neuronal Ceroid Lipofuscinosis

Recently, mutations in the DNAJC5 gene encoding cysteine-string protein α (CSPα) were identified to cause the neurodegenerative disorder adult-onset neuronal ceroid lipofuscinosis. The disease-causing mutations (L115R or ΔL116) occur within the cysteine-string domain, a region of the protein that is post-translationally modified by extensive palmitoylation. Here we demonstrate that L115R and ΔL116 mutant proteins are mistargeted in neuroendocrine cells and form SDS-resistant aggregates, concordant with the properties of other mutant proteins linked to neurodegenerative disorders. The mutant aggregates are membrane-associated and incorporate palmitate. Indeed, co-expression of palmitoyltransferase enzymes promoted the aggregation of the CSPα mutants, and chemical depalmitoylation solubilized the aggregates, demonstrating that aggregation is induced and maintained by palmitoylation. In agreement with these observations, SDS-resistant CSPα aggregates were present in brain samples from patients carrying the L115R mutation and were depleted by chemical depalmitoylation. In summary, this study identifies a novel interplay between genetic mutations and palmitoylation in driving aggregation of CSPα mutant proteins. We propose that this palmitoylation-induced aggregation of mutant CSPα proteins may underlie the development of adult-onset neuronal ceroid lipofuscinosis in affected families.     

Subcellular Localization Determines the Stability and Axon Protective Capacity of Axon Survival Factor Nmnat2

Axons require a constant supply of the labile axon survival factor Nmnat2 from their cell bodies to avoid spontaneous axon degeneration. Here we investigate the mechanism of fast axonal transport of Nmnat2 and its site of action for axon maintenance. Using dual-colour live-cell imaging of axonal transport in SCG primary culture neurons, we find that Nmnat2 is bidirectionally trafficked in axons together with markers of the trans-Golgi network and synaptic vesicles. In contrast, there is little co-migration with mitochondria, lysosomes, and active zone precursor vesicles. Residues encoded by the small, centrally located exon 6 are necessary and sufficient for stable membrane association and vesicular axonal transport of Nmnat2. Within this sequence, a double cysteine palmitoylation motif shared with GAP43 and surrounding basic residues are all required for efficient palmitoylation and stable association with axonal transport vesicles. Interestingly, however, disrupting this membrane association increases the ability of axonally localized Nmnat2 to preserve transected neurites in primary culture, while re-targeting the strongly protective cytosolic mutants back to membranes abolishes this increase. Larger deletions within the central domain including exon 6 further enhance Nmnat2 axon protective capacity to levels that exceed that of the slow Wallerian degeneration protein, Wld^S. The mechanism underlying the increase in axon protection appears to involve an increased half-life of the cytosolic forms, suggesting a role for palmitoylation and membrane attachment in Nmnat2 turnover. We conclude that Nmnat2 activity supports axon survival through a site of action distinct from Nmnat2 transport vesicles and that protein stability, a key determinant of axon protection, is enhanced by mutations that disrupt palmitoylation and dissociate Nmnat2 from these vesicles.     

Drosophila NMNAT maintains neural integrity independent of its NAD synthesis activity

Wallerian degeneration refers to a loss of the distal part of an axon after nerve injury. Wallerian degeneration slow (Wld^s) mice overexpress a chimeric protein containing the NAD synthase NMNAT (nicotinamide mononucleotide adenylyltransferase 1) and exhibit a delay in axonal degeneration. Currently, conflicting evidence raises questions as to whether NMNAT is the protecting factor and whether its enzymatic activity is required for such a possible function. Importantly, the link between nmnat and axon degeneration is at present solely based on overexpression studies of enzymatically active protein. Here we use the visual system of Drosophila as a model system to address these issues. We have isolated the first nmnat mutations in a multicellular organism in a forward genetic screen for synapse malfunction in Drosophila. Loss of nmnat causes a rapid and severe neurodegeneration that can be attenuated by blocking neuronal activity. Furthermore, in vivo neuronal expression of mutated nmnat shows that enzymatically inactive NMNAT protein retains strong neuroprotective effects and rescues the degeneration phenotype caused by loss of nmnat. Our data indicate an NAD-independent requirement of NMNAT for maintaining neuronal integrity that can be exploited to protect neurons from neuronal activity-induced degeneration by overexpression of the protein.     

Mutations in the X-linked Intellectual Disability Gene,zDHHC9, Alter Autopalmitoylation Activity by Distinct Mechanisms

Early onset intellectual disabilities result in significant societal and economic costs and affect 1–3% of the population. The underlying genetic determinants are beginning to emerge and are interpreted in the context of years of work characterizing postsynaptic receptor and signaling functions of learning and memory. DNA sequence analysis of intellectual disability patients has revealed greater than 80 loci on the X-chromosome that are potentially linked to disease. One of the loci is zDHHC9, a gene encoding a Ras protein acyltransferase. Protein palmitoylation is a reversible modification that controls the subcellular localization and distribution of membrane receptors, scaffolds, and signaling proteins required for neuronal plasticity. Palmitoylation occurs in two steps. In the first step, autopalmitoylation, an enzyme-palmitoyl intermediate is formed. During the second step, the palmitoyl moiety is transferred to a protein substrate, or if no substrate is available, hydrolysis of the thioester linkage produces the enzyme and free palmitate. In this study, we demonstrate that two naturally occurring variants of zDHHC9, encoding R148W and P150S, affect the autopalmitoylation step of the reaction by lowering the steady state amount of the palmitoyl-zDHHC9 intermediate.     

Protein depalmitoylases

Protein depalmitoylation describes the removal of thioester-linked long chain fatty acids from cysteine residues in proteins. For many S-palmitoylated proteins, this process is promoted by acyl protein thioesterase enzymes, which catalyze thioester hydrolysis to solubilize and displace substrate proteins from membranes. The closely related enzymes acyl protein thioesterase 1 (APT1; LYPLA1) and acyl protein thioesterase 2 (APT2; LYPLA2) were initially identified from biochemical assays as G protein depalmitoylases, yet later were shown to accept a number of S-palmitoylated protein and phospholipid substrates. Leveraging the development of isoform-selective APT inhibitors, several studies report distinct roles for APT enzymes in growth factor and hormonal signaling. Recent crystal structures of APT1 and APT2 reveal convergent acyl binding channels, suggesting additional factors beyond acyl chain recognition mediate substrate selection. In addition to APT enzymes, the ABHD17 family of hydrolases contributes to the depalmitoylation of Ras-family GTPases and synaptic proteins. Overall, enzymatic depalmitoylation ensures efficient membrane targeting by balancing the palmitoylation cycle, and may play additional roles in signaling, growth, and cell organization. In this review, we provide a perspective on the biochemical, structural, and cellular analysis of protein depalmitoylases, and outline opportunities for future studies of systems-wide analysis of protein depalmitoylation.     

2-Bromopalmitate Reduces Protein Deacylation by Inhibition of Acyl-Protein Thioesterase Enzymatic Activities

S-acylation, the covalent attachment of palmitate and other fatty acids on cysteine residues, is a reversible post-translational modification that exerts diverse effects on protein functions. S-acylation is catalyzed by protein acyltransferases (PAT), while deacylation requires acyl-protein thioesterases (APT), with numerous inhibitors for these enzymes having already been developed and characterized. Among these inhibitors, the palmitate analog 2-brompalmitate (2-BP) is the most commonly used to inhibit palmitoylation in cells. Nevertheless, previous results from our laboratory have suggested that 2-BP could affect protein deacylation. Here, we further investigated in vivo and in vitro the effect of 2-BP on the acylation/deacylation protein machinery, with it being observed that 2-BP, in addition to inhibiting PAT activity in vivo, also perturbed the acylation cycle of GAP-43 at the level of depalmitoylation and consequently affected its kinetics of membrane association. Furthermore, 2-BP was able to inhibit in vitro the enzymatic activities of human APT1 and APT2, the only two thioesterases shown to mediate protein deacylation, through an uncompetitive mechanism of action. In fact, APT1 and APT2 hydrolyzed both the monomeric form as well as the micellar state of the substrate palmitoyl-CoA. On the basis of the obtained results, as APTs can mediate deacylation on membrane bound and unbound substrates, this suggests that the access of APTs to the membrane interface is not a necessary requisite for deacylation. Moreover, as the enzymatic activity of APTs was inhibited by 2-BP treatment, then the kinetics analysis of protein acylation using 2-BP should be carefully interpreted, as this drug also inhibits protein deacylation.     

MEK Inhibitor U0126 Reverses Protection of Axons from Wallerian Degeneration Independently of MEK–ERK Signaling

Wallerian degeneration is delayed when sufficient levels of proteins with NMNAT activity are maintained within axons after injury. This has been proposed to form the basis of ''slow Wallerian degeneration'' (Wld ^S), a neuroprotective phenotype conferred by an aberrant fusion protein, Wld^S. Proteasome inhibition also delays Wallerian degeneration, although much less robustly, with stabilization of NMNAT2 likely to play a key role in this mechanism. The pan-MEK inhibitor U0126 has previously been shown to reverse the axon-protective effects of proteasome inhibition, suggesting that MEK-ERK signaling plays a role in delayed Wallerian degeneration, in addition to its established role in promoting neuronal survival. Here we show that whilst U0126 can also reverse Wld^S-mediated axon protection, more specific inhibitors of MEK1/2 and MEK5, PD184352 and BIX02189, have no significant effect on the delay to Wallerian degeneration in either situation, whether used alone or in combination. This suggests that an off-target effect of U0126 is responsible for reversion of the axon protective effects of Wld^S expression or proteasome inhibition, rather than inhibition of MEK1/2-ERK1/2 or MEK5-ERK5 signaling. Importantly, this off-target effect does not appear to result in alterations in the stabilities of either Wld^S or NMNAT2.     

Characterization of a Serine Hydrolase Targeted by Acyl-protein Thioesterase Inhibitors in Toxoplasma gondii

In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of β-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the β-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein.     

DHHC5 protein palmitoylates flotillin-2 and is rapidly degraded on induction of neuronal differentiation in cultured cells

Post-translational palmitoylation of intracellular proteins is mediated by protein palmitoyltransferases belonging to the DHHC family, which share a common catalytic Asp-His-His-Cys (DHHC) motif. Several members have been implicated in neuronal development, neurotransmission, and synaptic plasticity. We previously observed that mice homozygous for a hypomorphic allele of the ZDHHC5 gene are impaired in context-dependent learning and memory. To identify potentially relevant protein substrates of DHHC5, we performed a quantitative proteomic analysis of stable isotope-labeled neuronal stem cell cultures from forebrains of normal and DHHC5-GT (gene-trapped) mice using the bioorthogonal palmitate analog 17-octadecynoic acid. We identified ～300 17-octadecynoic acid-modified and hydroxylamine-sensitive proteins, of which a subset was decreased in abundance in DHHC5-GT cells. Palmitoylation and oligomerization of one of these proteins (flotillin-2) was abolished in DHHC5-GT neuronal stem cells. In COS-1 cells, overexpression of DHHC5 markedly stimulated the palmitoylation of flotillin-2, strongly suggesting a direct enzyme-substrate relationship. Serendipitously, we found that down-regulation of DHHC5 was triggered within minutes following growth factor withdrawal from normal neural stem cells, a maneuver that is used to induce neural differentiation in culture. The effect was reversible for up to 4 h, and degradation was partially prevented by inhibitors of ubiquitin-mediated proteolysis. These findings suggest that protein palmitoylation can be regulated through changes in DHHC PAT levels in response to differentiation signals.     

A fluorescence-based high performance liquid chromatographic method for the characterization of palmitoyl acyl transferase activity

Although protein palmitoylation is essential for targeting many important signaling proteins to the plasma membrane, the mechanism by which palmitoylation occurs is uncharacterized, since the enzyme(s) responsible for this modification remain unidentified. To study palmitoyl acyl transferase (PAT) activity, we developed an in vitro palmitoylation (IVP) assay using a fluorescently labeled substrate peptide, mimicking the N-terminal palmitoylation motif of proteins such as non-receptor Src-related tyrosine kinases. The palmitoylated and non-palmitoylated forms of the peptide were resolved by reverse-phase HPLC and detected by fluorescence. The method was optimized for PAT activity using lysates from the MCF-7 and Hep-G2 human tumor cell lines. The PAT activity was inhibited by boiling, reducing the incubation temperature, or adding 10 microM 2-bromopalmitate, a known palmitoylation inhibitor. This IVP assay provides the first method that is suitable to study all facets of the palmitoylation reaction, including peptide palmitoylation by PAT(s), depalmitoylation by thioesterases, and evaluation of potential palmitoylation inhibitors.     

In Silico Screening for Palmitoyl Substrates Reveals a Role for DHHC1/3/10 (zDHHC1/3/11)-mediated Neurochondrin Palmitoylation in Its Targeting to Rab5-positive Endosomes

Protein palmitoylation, a common post-translational lipid modification, plays an important role in protein trafficking and functions. Recently developed palmitoyl-proteomic methods identified many novel substrates. However, the whole picture of palmitoyl substrates has not been clarified. Here, we performed global in silico screening using the CSS-Palm 2.0 program, free software for prediction of palmitoylation sites, and selected 17 candidates as novel palmitoyl substrates. Of the 17 candidates, 10 proteins, including 6 synaptic proteins (Syd-1, transmembrane AMPA receptor regulatory protein (TARP) γ-2, TARP γ-8, cornichon-2, Ca²⁺/calmodulin-dependent protein kinase IIα, and neurochondrin (Ncdn)/norbin), one focal adhesion protein (zyxin), two ion channels (TRPM8 and TRPC1), and one G-protein-coupled receptor (orexin 2 receptor), were palmitoylated. Using the DHHC palmitoylating enzyme library, we found that all tested substrates were palmitoylated by the Golgi-localized DHHC3/7 subfamily. Ncdn, a regulator for neurite outgrowth and synaptic plasticity, was robustly palmitoylated by the DHHC1/10 (zDHHC1/11; z1/11) subfamily, whose substrate has not yet been reported. As predicted by CSS-Palm 2.0, Cys-3 and Cys-4 are the palmitoylation sites for Ncdn. Ncdn was specifically localized in somato-dendritic regions, not in the axon of rat cultured neurons. Stimulated emission depletion microscopy revealed that Ncdn was localized to Rab5-positive early endosomes in a palmitoylation-dependent manner, where DHHC1/10 (z1/11) were also distributed. Knockdown of DHHC1, -3, or -10 (z11) resulted in the loss of Ncdn from Rab5-positive endosomes. Thus, through in silico screening, we demonstrate that Ncdn and the DHHC1/10 (z1/11) and DHHC3/7 subfamilies are novel palmitoyl substrate-enzyme pairs and that Ncdn palmitoylation plays an essential role in its specific endosomal targeting.     

The Autodepalmitoylating Activity of APT Maintains the Spatial Organization of Palmitoylated Membrane Proteins

The localization and signaling of S-palmitoylated peripheral membrane proteins is sustained by an acylation cycle in which acyl protein thioesterases (APTs) depalmitoylate mislocalized palmitoylated proteins on endomembranes. However, the APTs are themselves reversibly S-palmitoylated, which localizes thioesterase activity to the site of the antagonistc palmitoylation activity on the Golgi. Here, we resolve this conundrum by showing that palmitoylation of APTs is labile due to autodepalmitoylation, creating two interconverting thioesterase pools: palmitoylated APT on the Golgi and depalmitoylated APT in the cytoplasm, with distinct functionality. By imaging APT-substrate catalytic intermediates, we show that it is the depalmitoylated soluble APT pool that depalmitoylates substrates on all membranes in the cell, thereby establishing its function as release factor of mislocalized palmitoylated proteins in the acylation cycle. The autodepalmitoylating activity on the Golgi constitutes a homeostatic regulation mechanism of APT levels at the Golgi that ensures robust partitioning of APT substrates between the plasma membrane and the Golgi.     

The Autodepalmitoylating Activity of APT Maintains the Spatial Organization of Palmitoylated Membrane Proteins

The localization and signaling of S-palmitoylated peripheral membrane proteins is sustained by an acylation cycle in which acyl protein thioesterases (APTs) depalmitoylate mislocalized palmitoylated proteins on endomembranes. However, the APTs are themselves reversibly S-palmitoylated, which localizes thioesterase activity to the site of the antagonistc palmitoylation activity on the Golgi. Here, we resolve this conundrum by showing that palmitoylation of APTs is labile due to autodepalmitoylation, creating two interconverting thioesterase pools: palmitoylated APT on the Golgi and depalmitoylated APT in the cytoplasm, with distinct functionality. By imaging APT-substrate catalytic intermediates, we show that it is the depalmitoylated soluble APT pool that depalmitoylates substrates on all membranes in the cell, thereby establishing its function as release factor of mislocalized palmitoylated proteins in the acylation cycle. The autodepalmitoylating activity on the Golgi constitutes a homeostatic regulation mechanism of APT levels at the Golgi that ensures robust partitioning of APT substrates between the plasma membrane and the Golgi.     

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.