S-acylation of p62 promotes p62 droplet recruitment into autophagosomes in mammalian autophagy.

p62 is a well-characterized autophagy receptor that recognizes and sequesters specific cargoes into autophagosomes for degradation. p62 promotes the assembly and removal of ubiquitinated proteins by forming p62-liquid droplets. However, it remains unclear how autophagosomes efficiently sequester p62 droplets. Herein, we report that p62 undergoes reversible S-acylation in multiple human-, rat-, and mouse-derived cell lines, catalyzed by zinc-finger Asp-His-His-Cys S-acyltransferase 19 (ZDHHC19) and deacylated by acyl protein thioesterase 1 (APT1). S-acylation of p62 enhances the affinity of p62 for microtubule-associated protein 1 light chain 3 (LC3)-positive membranes and promotes autophagic membrane localization of p62 droplets, thereby leading to the production of small LC3-positive p62 droplets and efficient autophagic degradation of p62-cargo complexes. Specifically, increasing p62 acylation by upregulating ZDHHC19 or by genetic knockout of APT1 accelerates p62 degradation and p62-mediated autophagic clearance of ubiquitinated proteins. Thus, the protein S-acylation-deacylation cycle regulates p62 droplet recruitment to the autophagic membrane and selective autophagic flux, thereby contributing to the control of selective autophagic clearance of ubiquitinated proteins.

APT1-Mediated Depalmitoylation Regulates Hippocampal Synaptic Plasticity.

Palmitoylation may be relevant to the processes of learning and memory, and even disorders, such as post-traumatic stress disorder and aging-related cognitive decline. However, underlying mechanisms of palmitoylation in these processes remain unclear. Herein, we used acyl-biotin exchange, coimmunoprecipitation and biotinylation assays, and behavioral and electrophysiological methods, to explore whether palmitoylation is required for hippocampal synaptic transmission and fear memory formation, and involved in functional modification of synaptic proteins, such as postsynapse density-95 (PSD-95) and glutamate receptors, and detected if depalmitoylation by specific enzymes has influence on glutamatergic synaptic plasticity. Our results showed that global palmitoylation level, palmitoylation of PSD-95 and glutamate receptors, postsynapse density localization of PSD-95, surface expression of AMPARs, and synaptic strength of cultured hippocampal neurons were all enhanced by TTX pretreatment, and these can be reversed by inhibition of palmitoylation with palmitoyl acyl transferases inhibitors, 2-bromopalmitate and N-(tert-butyl) hydroxylamine hydrochloride. Importantly, we also found that acyl-protein thioesterase 1 (APT1)-mediated depalmitoylation is involved in palmitoylation of PSD-95 and glutamatergic synaptic transmission. Knockdown of APT1, not protein palmitoyl thioesterase 1, with shRNA, or selective inhibition, significantly increased AMPAR-mediated synaptic strength, palmitoylation levels, and synaptic or surface expression of PSD-95 and AMPARs. Results from hippocampal tissues and fear-conditioned rats showed that palmitoylation is required for synaptic strengthening and fear memory formation. These results suggest that palmitoylation and APT1-mediated depalmitoylation have critical effects on the regulation of glutamatergic synaptic plasticity, and it may serve as a potential target for learning and memory-associated disorders.SIGNIFICANCE STATEMENT Fear-related anxiety disorders, including post-traumatic stress disorder, are prevalent psychiatric conditions, and fear memory is associated with hyperexcitability in the hippocampal CA1 region. Palmitoylation is involved in learning and memory, but mechanisms coupling palmitoylation with fear memory acquisition remain poorly understood. This study demonstrated that palmitoylation is essential for postsynapse density-95 clustering and hippocampal glutamatergic synaptic transmission, and APT1-mediated depalmitoylation plays critical roles in the regulation of synaptic plasticity. Our study revealed that molecular mechanism about downregulation of APT1 leads to enhancement of AMPAR-mediated synaptic transmission, and that palmitoylation cycling is implicated in fear conditioning-induced synaptic strengthening and fear memory formation.

Targeting the Ras palmitoylation/depalmitoylation cycle in cancer.

The Ras proteins are well-known drivers of many cancers and thus represent attractive targets for the development of anticancer therapeutics. Inhibitors that disrupt the association of the Ras proteins with membranes by blocking the addition of the farnesyl lipid moiety to the Ras C-terminus failed in clinical trials. Here, we explore the possibility of targeting a second lipid modification, S-acylation, commonly referred to as palmitoylation, as a strategy to disrupt the membrane interaction of specific Ras isoforms. We review the enzymes involved in adding and removing palmitate from Ras and discuss their potential roles in regulating Ras tumorigenesis. In addition, we examine other proteins that affect Ras protein localization and may serve as future drug targets.

Identification of palmitoyltransferase and thioesterase enzymes that control the subcellular localization of axon survival factor nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2).

The NAD-synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) is a critical survival factor for axons and its constant supply from neuronal cell bodies into axons is required for axon survival in primary culture neurites and axon extension in vivo. Recently, we showed that palmitoylation is necessary to target NMNAT2 to post-Golgi vesicles, thereby influencing its protein turnover and axon protective capacity. Here we find that NMNAT2 is a substrate for cytosolic thioesterases APT1 and APT2 and that palmitoylation/depalmitoylation dynamics are on a time scale similar to its short half-life. Interestingly, however, depalmitoylation does not release NMNAT2 from membranes. The mechanism of palmitoylation-independent membrane attachment appears to be mediated by the same minimal domain required for palmitoylation itself. Furthermore, we identify several zDHHC palmitoyltransferases that influence NMNAT2 palmitoylation and subcellular localization, among which a role for zDHHC17 (HIP14) in neuronal NMNAT2 palmitoylation is best supported by our data. These findings shed light on the enzymatic regulation of NMNAT2 palmitoylation and highlight individual thioesterases and palmitoyltransferases as potential targets to modulate NMNAT2-dependent axon survival.

Caveolin-2 palmitoylation turnover facilitates insulin receptor substrate-1-directed lipid metabolism by insulin receptor tyrosine kinase.

Here, we show that insulin induces palmitoylation turnover of Caveolin-2 (Cav-2) in adipocytes. Acyl protein thioesterases-1 (APT1) catalyzes Cav-2 depalmitoylation, and zinc finger DHHC domain-containing protein palmitoyltransferase 21 (ZDHHC21) repalmitoylation of the depalmitoylated Cav-2 for the turnover, thereby controlling insulin receptor (IR)-Cav-2-insulin receptor substrate-1 (IRS-1)-Akt-driven signaling. Insulin-induced palmitoylation turnover of Cav-2 facilitated glucose uptake and fat storage through induction of lipogenic genes. Cav-2-, APT1-, and ZDHHC21-deficient adipocytes, however, showed increased induction of lipolytic genes and glycerol release. In addition, white adipose tissues from insulin sensitive and resistant obese patients exhibited augmented expression of LYPLA1 (APT1) and ZDHHC20 (ZDHHC20). Our study identifies the specific enzymes regulating Cav-2 palmitoylation turnover, and reveals a new mechanism by which insulin-mediated lipid metabolism is controlled in adipocytes.

Deacylases-structure, function, and relationship to diseases.

Reversible S-acylation plays a pivotal role in various biological processes, modulating protein functions such as subcellular localization, protein stability/activity, and protein-protein interactions. These modifications are mediated by acyltransferases and deacylases, among which the most abundant modification is S-palmitoylation. Growing evidence has shown that this rivalrous pair of modifications, occurring in a reversible cycle, is essential for various biological functions. Aberrations in this process have been associated with various diseases, including cancer, neurological disorders, and immune diseases. This underscores the importance of studying enzymes involved in acylation and deacylation to gain further insights into disease pathogenesis and provide novel strategies for disease treatment. In this Review, we summarize our current understanding of the structure and physiological function of deacylases, highlighting their pivotal roles in pathology. Our aim is to provide insights for further clinical applications.

S-acylation regulates SQSTM1/p62-mediated selective autophagy.

Macroautophagy/autophagy is a highly conserved metabolic process that degrades intracellular components and recycles bioenergetic substrates. SQSTM1/p62 (sequestosome 1) is a classical autophagy receptor that participates in selective autophagy to eliminate abnormal intracellular components and recycle bioenergetic substrates. In autophagy, SQSTM1 recruits ubiquitinated substrates to form SQSTM1 droplets and delivers these cargoes to phagophores, the precursors to autophagosomes. Recently, we reported a previously unidentified SQSTM1 S-acylation, which is catalyzed by S-acyltransferase ZDHHC19 and reversed by LYPLA1/APT1. S-acylation of SQSTM1 enhances the affinity of SQSTM1 droplets with the phagophore membrane, thereby promoting efficient autophagic degradation of ubiquitinated substrates. Our study uncovers the role of the S-acylation-deacylation cycle in regulating SQSTM1-mediated selective autophagy.

Acyl-protein thioesterase1 alleviates senile osteoporosis by promoting osteoblast differentiation via depalmitoylation of BMPR1a.

Objective: Senile osteoporosis (SOP) is an aging-related disease. The depalmitoylating enzyme Acyl-protein thiesterase1 (APT1) is involved in disease regulation. This study explored the mechanism of APT1 in SOP. Methods: Eight-week-old SAMP6 mice were selected as SOP models and SAMR1 mice were controls, while osteoblasts were isolated from the femoral surface-soft tissues of SOP and control mice as in vitro models. Mouse femur morphological, bone mineral density (BMD), femur maximum elastic stress and maximum load, and APT1 expression were detected by HE staining, X-ray bone densitometer, material testing machine, and RT-qPCR and Western blot (WB). Osteoprotegrin (OPG)-labeled osteoblasts and APT1 localization in bone tissues were detected by immunohistochemical staining. APT1 expression was promoted in SOP mice by tail vein injection of APT1 lentivirus or promoted/silenced in osteoblasts by transfection of pcDNA3.1-APT1 overexpression or si-APT1 plasmids. SOP mouse osteoblast differentiation (OD), OD-related protein levels, osteoblast proliferation, BMPR1a palmitoylation level, and BMP/Smad pathway were detected by alizarin red staining, ALP activity detection, WB, CCK-8, and IP-ABE method. The effects of the pathway inhibitor LDN-193189 on OD were detected. Results: APT1 was under-expressed in osteoblasts of bone tissue in SOP mice and mainly localized in osteoblasts. SOP mice manifested increased bone marrow cavity and bone trabecular space, thinned trabecular bone, decreased BMD, maximum elastic stress, maximum load, and reduced OPG-positive osteoblasts in bone tissues, which were averted by APT1 overexpression, thus alleviating SOP. APT1 overexpression increased osteoblast calcium nodules, ALP activity, OD-related protein levels, and cell proliferation. In mechanism, APT1 overexpression inhibited BMPR1a palmitoylation in SOP mouse osteoblasts and activated the BMP/Smad pathway, thus promoting OD. Conclusion: APT1 activated the BMP/Smad pathway and promoted OD by regulating BMPR1a depalmitoylation, thus alleviating mouse SOP.

Ghrelin Acylation-A Post-Translational Tuning Mechanism Regulating Adult Hippocampal Neurogenesis.

Adult hippocampal neurogenesis-the generation of new functional neurones in the adult brain-is impaired in aging and many neurodegenerative disorders. We recently showed that the acylated version of the gut hormone ghrelin (acyl-ghrelin) stimulates adult hippocampal neurogenesis while the unacylated form of ghrelin inhibits it, thus demonstrating a previously unknown function of unacyl-ghrelin in modulating hippocampal plasticity. Analysis of plasma samples from Parkinson's disease patients with dementia demonstrated a reduced acyl-ghrelin:unacyl-ghrelin ratio compared to both healthy controls and cognitively intact Parkinson's disease patients. These data, from mouse and human studies, suggest that restoring acyl-ghrelin signalling may promote the activation of pathways to support memory function. In this short review, we discuss the evidence for ghrelin's role in regulating adult hippocampal neurogenesis and the enzymes involved in ghrelin acylation and de-acylation as targets to treat mood-related disorders and dementia.

Endothelial-Derived APT1-Mediated Macrophage-Endothelial Cell Interactions Participate in the Development of Atherosclerosis by Regulating the Ras/MAPK Signaling Pathway.

Acyl-protein thioesterase 1 (APT1) can affect H-Ras localization and function by promoting its depalmitoylation. However, relatively little attention has been paid to the effects of APT1 on H-Ras in the cardiovascular system. In this study, we revealed its roles in atherosclerosis development using oxidative low-density lipoprotein (ox-LDL)-induced endothelial dysfunction models and a Western diet-induced ApoE−/− mouse model. The results showed that APT1 expression was up-regulated, while that of miR-138-5p (miR-138) was down-regulated (p < 0.05) in this model. In the meantime, APT1 and H-Ras were translocated from the cytoplasm to the plasma membrane. Bioinformatic analysis and double fluorescence identified miR-138 as the upstream regulator of APT1. APT1 knockdown regulated H-Ras localization and expression, which subsequently affected the MAPK signaling pathway and the expression of its downstream factors. Further research indicated that human umbilical vein endothelial cells (HUVECs)-derived biogenic nanoparticles (BiNPs), hBPs secretion, and RNA expression of hBP-loaded APT1 were increased (p < 0.05) in the ox-LDL induced endothelial dysfunction model. Meanwhile, the HUVECs-derived APT1 could further affect macrophage function through hBP transportation. Altogether, this study demonstrated that the miR-138-APT1 axis may be partially responsible for atherosclerosis development by regulating the H-Ras-MAPK signaling pathway and hBP transportation. The results also shed novel insight on the underlying mechanisms of, and identify potential diagnostic and therapeutic targets for, atherosclerotic cardiovascular diseases in the future.

Dual-Drug Loaded Separable Microneedles for Efficient Rheumatoid Arthritis Therapy.

Although the inhibitors of the interleukin-6 receptor (IL-6R) and tumor necrosis factor-α (TNF-α) have achieved a certain success in the clinical treatment of rheumatoid arthritis (RA), great effort should be made to overcome side effects and to improve patient compliance. The present research aimed to address these problems by the co-delivery of tocilizumab (TCZ)-an inhibitor of IL-6R-and an aptamer Apt1-67, which specifically inhibits TNF receptor 1 via separable microneedles (MN). MN were featured with a sustained release of TCZ from needle tips and a rapid release of Apt1-67 from needle bodies by using methacrylate groups grafted hyaluronic acid as the fillings of needle tips and polyvinyl alcohol/polyvinyl pyrrolidone as the fillings of needle bodies. Our results demonstrated that TCZ and Apt1-67 were distributed in MN as expected, and they could be released to the surroundings in the skin. In vivo studies revealed that combined medication via MN (TCZ/Apt1-67@MN) was superior to MN loaded with a single drug. Compared with subcutaneous injection, TCZ/Apt1-67@MN was of great advantage in inhibiting bone erosion and alleviating symptoms of CIA mice. This study not only provides a novel approach for combined medication with different release properties but also supplies a strategy for improving drug efficacy.

Insights into the molecular basis of the palmitoylation and depalmitoylation of NCX1.

Catalyzed by zDHHC-PAT enzymes and reversed by thioesterases, protein palmitoylation is the only post-translational modification recognized to regulate the sodium/calcium exchanger NCX1. NCX1 palmitoylation occurs at a single site at position 739 in its large regulatory intracellular loop. An amphipathic ɑ-helix between residues 740-756 is a critical for NCX1 palmitoylation. Given the rich background of the structural elements involving in NCX1 palmitoylation, the molecular basis of NCX1 palmitoylation is still relatively poorly understood. Here we found that (1) the identity of palmitoylation machinery of NCX1 controls its spatial organization within the cell, (2) the NCX1 amphipathic ɑ-helix directly interacts with zDHHC-PATs, (3) NCX1 is still palmitoylated when it is arrested in either Golgi or ER, indicating that NCX1 is a substrate for multiple zDHHC-PATs, (4) the thioesterase APT1 but not APT2 as a part of NCX1-depalmitoylation machinery governs subcellular organization of NCX1, (5) APT1 catalyzes NCX1 depalmitoylation in the Golgi but not in the ER. We also report that NCX2 and NCX3 are dually palmitoylated, with important implications for substrate recognition and enzyme catalysis by zDHHC-PATs. Our results could support new molecular or pharmacological strategies targeting the NCX1 palmitoylation and depalmitoylation machinery.

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.

The binding of the APT1 domains to phosphoinositides is regulated by metal ions in vitro.

Chorein is a protein of the Vps13 family, and defects in this protein cause the rare neurodegenerative disorder chorea-acanthocytosis (ChAc). Chorein is involved in the actin cytoskeleton organization, calcium ion flux, neuronal cell excitability, exocytosis and autophagy. The function of this protein is poorly understood, and obtaining this knowledge is a key to finding a cure for ChAc. Chorein, as well as the Vps13 protein from yeast, contains the APT1 domain. Our previous research has shown that the APT1 domain from yeast Vps13 (yAPT1v) binds phosphatidylinositol 3-phosphate (PI3P) in vitro. In this study, we showed that although the APT1 domain from chorein (hAPT1) binds to PI3P it could not functionally replace yAPT1v. The hAPT1 domain binds, in addition to PI3P, to phosphatidylinositol 5-phosphate (PI5P). The binding of hAPT1 to PI3P, unlike the binding of yAPT1v to PI3P, is regulated by the bivalent ions, calcium and magnesium. Regulation of PI3P binding via calcium is also observed for the APT1 domain of yeast autophagy protein Atg2. The substitution I2771R, found in chorein of patient suffering from ChAc, reduces the binding of the hAPT1 domain to PI3P and PI5P. These results suggest that the ability of APT1 domains to bind phosphoinositides is regulated differently in yeast and human protein and that this regulation is important for chorein function.

RAS Subcellular Localization Inversely Regulates Thyroid Tumor Growth and Dissemination.

RAS mutations are the second most common genetic alteration in thyroid tumors. However, the extent to which they are associated with the most aggressive phenotypes is still controversial. Regarding their malignancy, the majority of RAS mutant tumors are classified as undetermined, which complicates their clinical management and can lead to undesired under- or overtreatment. Using the chick embryo spontaneous metastasis model, we herein demonstrate that the aggressiveness of HRAS-transformed thyroid cells, as determined by the ability to extravasate and metastasize at distant organs, is orchestrated by HRAS subcellular localization. Remarkably, aggressiveness inversely correlates with tumor size. In this respect, we also show that RAS site-specific capacity to regulate tumor growth and dissemination is dependent on VEGF-B secretion. Furthermore, we have identified the acyl protein thioesterase APT-1 as a determinant of thyroid tumor growth versus dissemination. We show that alterations in APT-1 expression levels can dramatically affect the behavior of thyroid tumors, based on its role as a regulator of HRAS sublocalization at distinct plasma membrane microdomains. In agreement, APT-1 emerges in thyroid cancer clinical samples as a prognostic factor. As such, APT-1 levels could serve as a biomarker that could help in the stratification of HRAS mutant thyroid tumors based on their aggressiveness.

Identifying the Potential Substrates of the Depalmitoylation Enzyme Acyl-protein Thioesterase 1.

BACKGROUND: The homeostasis of palmitoylation and depalmitoylation is involved in various cellular processes, the disruption of which induces severe physiological consequences. Acyl-protein thioesterase (APT) and palmitoyl-protein thioesterases (PPT) catalyze the depalmitoylation process. The natural mutation in human PPT1 caused neurodegenerative disease, yet the understanding of APT1 remains to be elucidated. While the deletion of APT1 in mice turned out to be potentially embryonically lethal, the decoding of its function strictly relied on the identification of its substrates. OBJECTIVE: To determine the potential substrates of APT1 by using the generated human APT1 knockout cell line. METHODS: The combined techniques of palmitoyl-protein enrichment and massspectrometry were used to analyze the different proteins. Palmitoyl-proteins both in HEK293T and APT1-KO cells were extracted by resin-assisted capture (RAC) and data independent acquisition (DIA) quantitative method of proteomics for data collection. RESULTS: In total, 382 proteins were identified. The gene ontology classification segregated these proteins into diverse biological pathways e.g. endoplasmic reticulum process and ubiquitin-mediated proteolysis. A few potential substrates were selected for verification; indeed, major proteins were palmitoylated. Importantly, their levels of palmitoylation were clearly changed in APT1-KO cells. Interestingly, the proliferation of APT1-KO cells escalated dramatically as compared to that of the WT cells, which could be rescued by APT1 overexpression. CONCLUSION: Our study provides a large scale of potential substrates of APT1, thus facilitating the understanding of its intervened molecular functions.

Acyl protein thioesterase 1 and 2 (APT-1, APT-2) inhibitors palmostatin B, ML348 and ML349 have different effects on NRAS mutant melanoma cells.

Oncogenic NRAS mutations are frequent in melanoma and lead to increased downstream signaling and uncontrolled cell proliferation. Since the direct inhibition of NRAS is not possible yet, modulators of NRAS posttranslational modifications have become an area of interest. Specifically, interfering with NRAS posttranslational palmitoylation/depalmitoylation cycle could disturb proper NRAS localization, and therefore decrease cell proliferation and downstream signaling. Here, we investigate the expression and function of NRAS depalmitoylating acyl protein thioesterases 1 and 2 (APT-1, APT-2) in a panel of NRAS mutant melanoma cells. First, we show that all melanoma cell lines examined express APT-1 and APT-2. Next, we show that siRNA mediated APT-1 and APT-2 knock down and that the specific APT-1 and -2 inhibitors ML348 and ML349 have no biologically significant effects in NRAS mutant melanoma cells. Finally, we test the dual APT-1 and APT-2 inhibitor palmostatin B and conclude that palmostatin B has effects on NRAS downstream signaling and cell viability in NRAS mutant melanoma cells, offering an interesting starting point for future studies.

ABHD17 proteins are novel protein depalmitoylases that regulate N-Ras palmitate turnover and subcellular localization.

Dynamic changes in protein S-palmitoylation are critical for regulating protein localization and signaling. Only two enzymes - the acyl-protein thioesterases APT1 and APT2 - are known to catalyze palmitate removal from cytosolic cysteine residues. It is unclear if these enzymes act constitutively on all palmitoylated proteins, or if additional depalmitoylases exist. Using a dual pulse-chase strategy comparing palmitate and protein half-lives, we found knockdown or inhibition of APT1 and APT2 blocked depalmitoylation of Huntingtin, but did not affect palmitate turnover on postsynaptic density protein 95 (PSD95) or N-Ras. We used activity profiling to identify novel serine hydrolase targets of the APT1/2 inhibitor Palmostatin B, and discovered that a family of uncharacterized ABHD17 proteins can accelerate palmitate turnover on PSD95 and N-Ras. ABHD17 catalytic activity is required for N-Ras depalmitoylation and re-localization to internal cellular membranes. Our findings indicate that the family of depalmitoylation enzymes may be substantially broader than previously believed.

S-palmitoylation regulates AMPA receptors trafficking and function: a novel insight into synaptic regulation and therapeutics.

Glutamate acting on AMPA-type ionotropic glutamate receptor (AMPAR) mediates the majority of fast excitatory synaptic transmission in the mammalian central nervous system. Dynamic regulation of AMPAR by post-translational modifications is one of the key elements that allow the nervous system to adapt to environment stimulations. S-palmitoylation, an important lipid modification by post-translational addition of a long-chain fatty acid to a cysteine residue, regulates AMPA receptor trafficking, which dynamically affects multiple fundamental brain functions, such as learning and memory. In vivo, S-palmitoylation is controlled by palmitoyl acyl transferases and palmitoyl thioesterases. In this review, we highlight advances in the mechanisms for dynamic AMPA receptors palmitoylation, and discuss how palmitoylation affects AMPA receptors function at synapses in recent years. Pharmacological regulation of S-palmitoylation may serve as a novel therapeutic strategy for neurobiological diseases.

Adenine phosphoribosyl transferase 1 is a key enzyme catalyzing cytokinin conversion from nucleobases to nucleotides in Arabidopsis.

In plants, the cytokinin metabolic processes, including cytokinin biosynthesis, interconversion, inactivation, and degradation, play critical roles in the regulation of cytokinin homeostasis and plant development. Purine metabolic enzymes have been implied to catalyze the cytokinin interconversion in previous works. In this study, we report that Adenine Phosphoribosyl Transferase 1 (APT1) is the causal gene of the high-dose cytokinin-resistant mutants. APT1 catalyzes the cytokinin conversion from free bases to nucleotides, and is functionally predominant among the five members of the Arabidopsis Adenine Phosphoribosyl Transferase family. Loss of APT1 activity in plants leads to excess accumulation of cytokinin bases, thus evoking myriad cytokinin-regulated responses, such as delayed leaf senescence, anthocyanin accumulation, and downstream gene expression. Thus, our study defines APT1 as a key metabolic enzyme participating in the cytokinin inactivation by phosphoribosylation.