Purification and characterization of recombinant protein acyltransferases

Palmitoylation enhances membrane association and plays a role in the subcellular trafficking and signaling function of proteins. Unlike other forms of protein lipidation, such as prenylation and myristoylation, palmitoylation is reversible and can therefore play a regulatory role. Enzyme activities have recently been described in mammals and yeast that carry out the palmitoylation of protein substrates. Protein acyltransferases (PATs) transfer a palmitoyl moiety derived from palmitoyl-CoA to a free thiol of a substrate protein to create a labile thioester linkage. Biochemical characterization and kinetic analysis of this new family of enzymes requires methods to purify PATs and their substrates, as well as methods to assay PAT activity. We describe a series of methods using yeast and bacterial expression systems to study protein acyltransferases.     

Palmitoylation by DHHC5/8 targets GRIP1 to dendritic endosomes to regulate AMPA-R trafficking

Palmitoylation, a key regulatory mechanism controlling protein targeting, is catalyzed by DHHC-family palmitoyl acyltransferases (PATs). Impaired PAT activity is linked to neurodevelopmental and neuropsychiatric disorders, suggesting critical roles for palmitoylation in neuronal function. However, few substrates for specific PATs are known, and functional consequences of palmitoylation events are frequently uncharacterized. Here, we identify the closely related PATs DHHC5 and DHHC8 as specific regulators of the PDZ domain protein GRIP1b. Binding, palmitoylation, and dendritic targeting of GRIP1b require a PDZ ligand unique to DHHC5/8. Palmitoylated GRIP1b is targeted to trafficking endosomes and may link endosomes to kinesin motors. Consistent with this trafficking role, GRIP1b's palmitoylation turnover rate approaches the highest of all reported proteins, and palmitoylation increases GRIP1b's ability to accelerate AMPA-R recycling. To our knowledge, these findings identify the first neuronal DHHC5/8 substrate, define novel mechanisms controlling palmitoylation specificity, and suggest further links between dysregulated palmitoylation and neuropathological conditions.     

Systematic screening for palmitoyl transferase activity of the DHHC protein family in mammalian cells

Posttranslational modifications, including phosphorylation, ubiquitination and lipid modifications, provide proteins with additional functions and regulation beyond genomic information. Palmitoylation is a reversible lipid modification with palmitic acid that plays critical roles in protein trafficking and function. However, the enzymes that mediate palmitoyl acyl transferase (PAT) have been elusive. Recent genetic analysis in yeast revealed that members of cysteine-rich DHHC domain containing proteins (DHHC proteins) mediate palmitoylation. In mammalian genomes, 23 DHHC proteins are predicted raising the possibility of a large family of PAT enzymes. Here, we describe a systematic method to examine which of the DHHC family members is responsible for palmitoylation of a substrate.     

Transmembrane topology of the protein palmitoyl transferase Akr1

The two recently identified protein acyl transferases (PATs), Akr1p and Erf2p/Erf4p, point toward the DHHC protein family as a likely PAT family. The DHHC protein family, defined by the novel, zinc finger-like DHHC cysteine-rich domain (DHHC-CRD), is a diverse collection of polytopic membrane proteins extending through all eukaryotes. To define the PAT domains that are oriented to the cytoplasm and are thus available to effect the cytoplasmically limited palmitoyl modification, we have determined the transmembrane topology of the yeast PAT Akr1p. Portions of the yeast protein invertase (Suc2p) were inserted in-frame at 10 different hydrophilic sites within the Akr1 polypeptide. Three of the Akr1-Suc2-Akr1 insertion proteins were found to be extensively glycosylated, indicating that the invertase segment inserted at these Akr1p sites is luminally oriented. The remaining seven insertion proteins were not glycosylated, consistent with a cytoplasmic orientation for these sites. The results support a model in which the Akr1 polypeptide crosses the bilayer six times with the bulk of its hydrophilic domains disposed toward the cytoplasm. Cytoplasmic domains include both the relatively large, ankyrin repeat-containing N-terminal domain and the DHHC-CRD, which maps to a cytosolic loop segment. Functionality of the different Akr1-Suc2-Akr1 proteins also was examined. Insertions at only 4 of the 10 sites were found to disrupt Akr1p function. Interestingly, these four sites all map cytoplasmically, suggesting key roles for these cytoplasmic domains in Akr1 PAT function. Finally, extrapolating from the Akr1p topology, topology models are proposed for other DHHC protein family members.     

DHHC palmitoyl transferases: substrate interactions and (patho)physiology

S-palmitoylation is a reversible post-translational modification that occurs on diverse cellular proteins. Palmitoylation can affect proteins in many different ways, including regulating membrane attachment, intracellular trafficking, and membrane micro-localisation. Intracellular palmitoylation reactions are mediated by a family of recently identified aspartate-histidine-histidine-cysteine (DHHC) palmitoyl transferases. More than 20 DHHC proteins are encoded by mammalian genomes, and there is now a major effort to identify DHHC-substrate pairings and to determine how interaction specificity is encoded. Recent studies have highlighted how DHHC proteins regulate cell function and influence physiology and pathophysiology.     

Palmitoylation cycles and regulation of protein function (Review)

The efficacy and success of many cellular processes is dependent on a tight orchestration of proteins trafficking to and from their site(s) of action in a time-controlled fashion. Recently, a dynamic cycle of palmitoylation/de-palmitoylation has been shown to regulate shuttling of several proteins, including the small GTPases H-Ras and N-Ras, and the GABA-synthesizing enzyme GAD65, between the Golgi compartment and either the plasma membrane or synaptic vesicle membranes. These proteins are peripheral membrane proteins that in the depalmitoylated state cycle rapidly on and off the cytosolic face of ER/Golgi membranes. Palmitoylation of one or more cysteines, by a Golgi localized palmitoyl transferase (PAT) results in trapping in Golgi membranes, and sorting to a vesicular pathway in route to the plasma membrane or synaptic vesicles. A depalmitoylation step by an acyl protein thioesterase (APT) releases the protein from membranes in the periphery of the cell resulting in retrograde trafficking back to Golgi membranes by a non-vesicular pathway. The proteins can then enter a new cycle of palmitoylation and depalmitoylation. This inter-compartmental trafficking is orders of magnitude faster than vesicular trafficking. Recent advances in identifying a large family of PATs, their protein substrates, and single PAT mutants with severe phenotypes, reveal their critical importance in development, synaptic transmission, and regulation of signaling cascades. The emerging knowledge of enzymes involved in adding and removing palmitate is that they provide an intricate regulatory network involved in timing of protein function and transport that responds to intracellular and extracellular signals.     

Palmitoylation of R-Ras by human DHHC19, a palmitoyl transferase with a CaaX box

Mammalian proteins that contain an aspartate-histidine-histidine-cysteine-(DHHC) motif have been recently identified as a group of membrane-associated palmitoyl acyltransferases (PATs). Among the several protein substrates known to become palmitoylated by DHHC PATs are small GTPases prenylated at their carboxy-terminal end, such as H-Ras or N-Ras, eNOS, kinases myristoylated at their N-terminal end, such as Lck, and many transmembrane proteins and channels. We have focused our studies on the product of the human gene DHHC19, a putative palmitoyl transferase that, interestingly, displays a conserved CaaX box at its carboxy-terminal end. We show herein that the amino acid sequence present at the carboxy-terminus of DHHC19 is able to exclude a green fluorescent protein (GFP) reporter from the nucleus and direct it towards perinuclear regions. Transfection of full-length DHHC19 in COS7 cells reveals a perinuclear distribution, in analogy to other palmitoyl transferases, with a strong colocalization with the trans-Golgi markers Gal-T and TGN38. We have tested several small GTPases that are known to be palmitoylated as possible substrates of DHHC19. Although DHHC19 failed to increase the palmitoylation of H-Ras, N-Ras, K-Ras4A, RhoB or Rap2 it increased the palmitoylation of R-Ras approximately two-fold. The increased palmitoylation of R-Ras cotransfected with DHHC19 is accompanied by an augmented association with membranes as well as with rafts/caveolae. Finally, using both wild-type and an activated GTP bound form of R-Ras (G38V), we also show that the increased palmitoylation of R-Ras due to DHHC19 coexpression is accompanied by an enhanced viability of the transfected cells.     

The Role of Palmitoylation for Protein Recruitment to the Inner Membrane Complex of the Malaria Parasite

To survive and persist within its human host, the malaria parasite Plasmodium falciparum utilizes a battery of lineage-specific innovations to invade and multiply in human erythrocytes. With central roles in invasion and cytokinesis, the inner membrane complex, a Golgi-derived double membrane structure underlying the plasma membrane of the parasite, represents a unique and unifying structure characteristic to all organisms belonging to a large phylogenetic group called Alveolata. More than 30 structurally and phylogenetically distinct proteins are embedded in the IMC, where a portion of these proteins displays N-terminal acylation motifs. Although N-terminal myristoylation is catalyzed co-translationally within the cytoplasm of the parasite, palmitoylation takes place at membranes and is mediated by palmitoyl acyltransferases (PATs). Here, we identify a PAT (PfDHHC1) that is exclusively localized to the IMC. Systematic phylogenetic analysis of the alveolate PAT family reveals PfDHHC1 to be a member of a highly conserved, apicomplexan-specific clade of PATs. We show that during schizogony this enzyme has an identical distribution like two dual-acylated, IMC-localized proteins (PfISP1 and PfISP3). We used these proteins to probe into specific sequence requirements for IMC-specific membrane recruitment and their interaction with differentially localized PATs of the parasite.     

Phosphatidylinositol 4-kinase IIα is palmitoylated by Golgi-localized palmitoyltransferases in cholesterol-dependent manner

Phosphatidylinositol 4-kinase IIα (PI4KIIα) is predominantly Golgi-localized, and it generates >50% of the phosphatidylinositol 4-phosphate in the Golgi. The lipid kinase activity, Golgi localization, and "integral" membrane binding of PI4KIIα and its association with low buoyant density "raft" domains are critically dependent on palmitoylation of its cysteine-rich (173)CCPCC(177) motif and are also highly cholesterol-dependent. Here, we identified the palmitoyl acyltransferases (Asp-His-His-Cys (DHHC) PATs) that palmitoylate PI4KIIα and show for the first time that palmitoylation is cholesterol-dependent. DHHC3 and DHHC7 PATs, which robustly palmitoylated PI4KIIα and were colocalized with PI4KIIα in the trans-Golgi network (TGN), were characterized in detail. Overexpression of DHHC3 or DHHC7 increased PI4KIIα palmitoylation by >3-fold, whereas overexpression of the dominant-negative PATs or PAT silencing by RNA interference decreased PI4KIIα palmitoylation, "integral" membrane association, and Golgi localization. Wild-type and dominant-negative DHHC3 and DHHC7 co-immunoprecipitated with PI4KIIα, whereas non-candidate DHHC18 and DHHC23 did not. The PI4KIIα (173)CCPCC(177) palmitoylation motif is required for interaction because the palmitoylation-defective SSPSS mutant did not co-immunoprecipitate with DHHC3. Cholesterol depletion and repletion with methyl-β-cyclodextrin reversibly altered PI4KIIα association with these DHHCs as well as PI4KIIα localization at the TGN and "integral" membrane association. Significantly, the Golgi phosphatidylinositol 4-phosphate level was altered in parallel with changes in PI4KIIα behavior. Our study uncovered a novel mechanism for the preferential recruitment and activation of PI4KIIα to the TGN by interaction with Golgi- and raft-localized DHHCs in a cholesterol-dependent manner.     

The Erf4 Subunit of the Yeast Ras Palmitoyl Acyltransferase Is Required for Stability of the Acyl-Erf2 Intermediate and Palmitoyl Transfer to a Ras2 Substrate

Protein S-palmitoylation is a posttranslational modification in which a palmitoyl group is added to a protein via a thioester linkage on cysteine. Palmitoylation is a reversible modification involved in protein membrane targeting, receptor trafficking and signaling, vesicular biogenesis and trafficking, protein aggregation, and protein degradation. An example of the dynamic nature of this modification is the palmitoylation-depalmitoylation cycle that regulates the subcellular trafficking of Ras family GTPases. The Ras protein acyltransferase (PAT) consists of a complex of Erf2-Erf4 and DHHC9-GCP16 in yeast and mammalian cells, respectively. Both subunits are required for PAT activity, but the function of the Erf4 and Gcp16 subunits has not been established. This study elucidates the function of Erf4 and shows that one role of Erf4 is to regulate Erf2 stability through an ubiquitin-mediated pathway. In addition, Erf4 is required for the stable formation of the palmitoyl-Erf2 intermediate, the first step of palmitoyl transfer to protein substrates. In the absence of Erf4, the rate of hydrolysis of the active site palmitoyl thioester intermediate is increased, resulting in reduced palmitoyl transfer to a Ras2 substrate. This is the first demonstration of regulation of a DHHC PAT enzyme by an associated protein.     

Cellular palmitoylation and trafficking of lipidated peptides

Many important signaling proteins require the posttranslational addition of fatty acid chains for their proper subcellular localization and function. One such modification is the addition of palmitoyl moieties by enzymes known as palmitoyl acyltransferases (PATs). Substrates for PATs include C-terminally farnesylated proteins, such as H- and N-Ras, as well as N-terminally myristoylated proteins, such as many Src-related tyrosine kinases. The molecular and biochemical characterization of PATs has been hindered by difficulties in developing effective methods for the analysis of PAT activity. In this study, we describe the use of cell-permeable, fluorescently labeled lipidated peptides that mimic the PAT recognition domains of farnesylated and myristoylated proteins. These PAT substrate mimetics are accumulated by SKOV3 cells in a saturable and time-dependent manner. Although both peptides are rapidly palmitoylated, the SKOV3 cells have a greater capacity to palmitoylate the myristoylated peptide than the farnesylated peptide. Confocal microscopy indicated that the palmitoylated peptides colocalized with Golgi and plasma membrane markers, whereas the corresponding nonpalmitoylatable peptides accumulated in the Golgi but did not traffic to the plasma membrane. Overall, these studies indicate that the lipidated peptides provide useful cellular probes for quantitative and compartmentalization studies of protein palmitoylation in intact cells.     

Protein palmitoylation by a family of DHHC protein S-acyltransferases

Protein palmitoylation refers to the posttranslational addition of a 16 carbon fatty acid to the side chain of cysteine, forming a thioester linkage. This acyl modification is readily reversible, providing a potential regulatory mechanism to mediate protein-membrane interactions and subcellular trafficking of proteins. The mechanism that underlies the transfer of palmitate or other long-chain fatty acids to protein was uncovered through genetic screens in yeast. Two related S-palmitoyltransferases were discovered. Erf2 palmitoylates yeast Ras proteins, whereas Akr1 modifies the yeast casein kinase, Yck2. Erf2 and Akr1 share a common sequence referred to as a DHHC (aspartate-histidine-histidine-cysteine) domain. Numerous genes encoding DHHC domain proteins are found in all eukaryotic genome databases. Mounting evidence is consistent with this signature motif playing a direct role in protein acyltransferase (PAT) reactions, although many questions remain. This review presents the genetic and biochemical evidence for the PAT activity of DHHC proteins and discusses the mechanism of protein-mediated palmitoylation.     

Identification of PSD-95 palmitoylating enzymes

Palmitoylation is a lipid modification that plays a critical role in protein trafficking and function throughout the nervous system. Palmitoylation of PSD-95 is essential for its regulation of AMPA receptors and synaptic plasticity. The enzymes that mediate palmitoyl acyl transfer to PSD-95 have not yet been identified; however, proteins containing a DHHC cysteine-rich domain mediate palmitoyl acyl transferase activity in yeast. Here, we isolated 23 mammalian DHHC proteins and found that a subset specifically palmitoylated PSD-95 in vitro and in vivo. These PSD-95 palmitoyl transferases (P-PATs) showed substrate specificity, as they did not all enhance palmitoylation of Lck, SNAP-25b, Galpha(s), or H-Ras in cultured cells. Inhibition of P-PAT activity in neurons reduced palmitoylation and synaptic clustering of PSD-95 and diminished AMPA receptor-mediated neurotransmission. This study suggests that P-PATs regulate synaptic function through PSD-95 palmitoylation.     

DHHC5 Mediates β-Adrenergic Signaling in Cardiomyocytes by Targeting Gα Proteins

S-palmitoylation is a reversible posttranslational modification that plays an important role in regulating protein localization, trafficking, and stability. Recent studies have shown that some proteins undergo extremely rapid palmitoylation/depalmitoylation cycles after cellular stimulation supporting a direct signaling role for this posttranslational modification. Here, we investigated whether β-adrenergic stimulation of cardiomyocytes led to stimulus-dependent palmitoylation of downstream signaling proteins. We found that β-adrenergic stimulation led to rapidly increased Gαs and Gαi palmitoylation. The kinetics of palmitoylation was temporally consistent with the downstream production of cAMP and contractile responses. We identified the plasma membrane-localized palmitoyl acyltransferase DHHC5 as an important mediator of the stimulus-dependent palmitoylation in cardiomyocytes. Knockdown of DHHC5 showed that this enzyme is necessary for palmitoylation of Gαs, Gαi, and functional responses downstream of β-adrenergic stimulation. A palmitoylation assay with purified components revealed that Gαs and Gαi are direct substrates of DHHC5. Finally, we provided evidence that the C-terminal tail of DHHC5 can be palmitoylated in response to stimulation and such modification is important for its dynamic localization and function in the plasma membrane. Our results reveal that DHHC5 is a central regulator of signaling downstream of β-adrenergic receptors in cardiomyocytes.     

Identification of CKAP4/p63 as a major substrate of the palmitoyl acyltransferase DHHC2, a putative tumor suppressor, using a novel proteomics method

Protein palmitoylation is the post-translational addition of the 16-carbon fatty acid palmitate to specific cysteine residues by a labile thioester linkage. Palmitoylation is mediated by a family of at least 23 palmitoyl acyltransferases (PATs) characterized by an Asp-His-His-Cys (DHHC) motif. Many palmitoylated proteins have been identified, but PAT-substrate relationships are mostly unknown. Here we present a method called palmitoyl-cysteine isolation capture and analysis (or PICA) to identify PAT-substrate relationships in a living vertebrate system and demonstrate its effectiveness by identifying CKAP4/p63 as a substrate of DHHC2, a putative tumor suppressor.     

Analysis of substrate specificity of human DHHC protein acyltransferases using a yeast expression system

Palmitoylation plays important roles in the regulation of protein localization, stability, and activity. The protein acyltransferases (PATs) have a common DHHC Cys-rich domain. Twenty-three DHHC proteins have been identified in humans. However, it is unclear whether all of these DHHC proteins function as PATs. In addition, their substrate specificities remain largely unknown. Here we develop a useful method to examine substrate specificities of PATs using a yeast expression system with six distinct model substrates. We identify 17 human DHHC proteins as PATs. Moreover, we classify 11 human and 5 yeast DHHC proteins into three classes (I, II, and III), based on the cellular localization of their respective substrates (class I, soluble proteins; class II, integral membrane proteins; class III, lipidated proteins). Our results may provide an important clue for understanding the function of individual DHHC proteins.     

Dynamic protein palmitoylation in cellular signaling

Protein S-palmitoylation, the most common lipid modification with the 16-carbon fatty acid palmitate, provides an important mechanism for regulating protein trafficking and function. The unique reversibility of protein palmitoylation allows proteins to rapidly shuttle between intracellular membrane compartments. Importantly, this palmitate cycling can be regulated by some physiological stimuli, contributing to cellular homeostasis and plasticity. Although the enzyme responsible for protein palmitoylation had been long elusive, DHHC family proteins, conserved from plants to mammals, have recently emerged as palmitoyl acyl transferases. Integrated approaches including advanced proteomics, live-cell imaging, and molecular genetics are beginning to clarify the molecular machinery for palmitoylation reaction in diverse aspects of cellular functions.     

Palmitoylation and membrane interactions of the neuroprotective chaperone cysteine-string protein

Cysteine-string protein (CSP) is an extensively palmitoylated DnaJ-family chaperone, which exerts an important neuroprotective function. Palmitoylation is required for the intracellular sorting and function of CSP, and thus it is important to understand how this essential modification of CSP is regulated. Recent work identified 23 putative palmitoyl transferases containing a conserved DHHC domain in mammalian cells, and here we show that palmitoylation of CSP is enhanced specifically by co-expression of the Golgi-localized palmitoyl transferases DHHC3, DHHC7, DHHC15, or DHHC17. Indeed, these DHHC proteins promote stable membrane attachment of CSP, which is otherwise cytosolic. An inverse correlation was identified between membrane affinity of unpalmitoylated CSP mutants and subsequent palmitoylation: mutants with an increased membrane affinity localize to the endoplasmic reticulum (ER) and are physically separated from the Golgi-localized DHHC proteins. Palmitoylation of an ER-localized mutant could be rescued by brefeldin A treatment, which promotes the mixing of ER and Golgi membranes. Interestingly though, the palmitoylated mutant remained at the ER following brefeldin A washout and did not traffic to more distal membrane compartments. We propose that CSP has a weak membrane affinity that allows the protein to locate its partner Golgi-localized DHHC proteins directly by membrane "sampling." Mutations that enhance membrane association prevent sampling and lead to accumulation of CSP on cellular membranes such as the ER. The coupling of CSP palmitoylation to Golgi membranes may thus be an important requirement for subsequent sorting.