The mitochondria-associated membrane (MAM) is a domain of the endoplasmic reticulum (ER) that mediates the exchange of ions, lipids and metabolites between the ER and mitochondria. ER chaperones and oxidoreductases are critical components of the MAM. However, the localization motifs and mechanisms for most MAM proteins have remained elusive. Using two highly related ER oxidoreductases as a model system, we now show that palmitoylation enriches ER-localized proteins on the MAM. We demonstrate that palmitoylation of cysteine residue(s) adjacent to the membrane-spanning domain promotes MAM enrichment of the transmembrane thioredoxin family protein TMX. In addition to TMX, our results also show that calnexin shuttles between the rough ER and the MAM depending on its palmitoylation status. Mutation of the TMX and calnexin palmitoylation sites and chemical interference with palmitoylation disrupt their MAM enrichment. Since ER-localized heme oxygenase-1, but not cytosolic GRP75 require palmitoylation to reside on the MAM, our findings identify palmitoylation as key for MAM enrichment of ER membrane proteins.

For lipid synthesis, energy production via ␤ -oxidation, or for protein fatty acylation to occur, long-chain fatty acids (LCFAs) must be activated by conversion to their CoA derivatives (LCFA-CoAs) by fatty acyl-CoA synthetase (FAS ). Protein fatty acylation is one of many types of posttranslational modifi cations of proteins by lipids, which also includes isoprenoids, glycosylphosphatidylinositols, and cholesterol. Typically, lipids covalently attached to proteins serve as hydrophobic membrane anchors ( 1-6 ).Protein fatty acylation is mainly divided into two categories: N-myristoylation and S-acylation. The corresponding reactions are catalyzed by N-myristoyl transferases (NMT1 and NMT2) and two families of protein acyltransferases (PATs) referred to as zinc fi nger, Asp-His-His-Cys PATs Abstract Progress in understanding the biology of protein fatty acylation has been impeded by the lack of rapid direct detection and identifi cation methods. We fi rst report that a synthetic -alkynyl-palmitate analog can be readily and specifi cally incorporated into GAPDH or mitochondrial 3-hydroxyl-3-methylglutaryl-CoA synthase in vitro and reacted with an azido-biotin probe or the fl uorogenic probe 3-azido-7-hydroxycoumarin using click chemistry for rapid detection by Western blotting or fl at bed fl uorescence scanning. The acylated cysteine residues were confi rmed by MS. Second, -alkynyl-palmitate is preferentially incorporated into transiently expressed H-or N-Ras proteins (but not nonpalmitoylated K-Ras), compared with -alkynyl-myristate or -alkynyl-stearate, via an alkali sensitive thioester bond. Third, -alkynyl-myristate is specifi cally incorporated into endogenous co-and posttranslationally myristoylated proteins. The competitive inhibitors 2-bromopalmitate and 2-hydroxymyristate prevented incorporation of -alkynylpalmitate and -alkynyl-myristate into palmitoylated and myristoylated proteins, respectively. Labeling cells with -alkynyl-palmitate does not affect membrane association of N-Ras. Furthermore, the palmitoylation of endogenous proteins including H-and N-Ras could be easily detected using -alkynyl-palmitate as label in cultured HeLa, Jurkat, and COS-7 cells, and, promisingly, in mice. The -alkynylmyristate and -palmitate analogs used with click chemistry

SummaryThe palmitoylation of calnexin serves to enrich calnexin on the mitochondria-associated membrane (MAM). Given a lack of information on the significance of this finding, we have investigated how this endoplasmic reticulum (ER)-internal sorting signal affects the functions of calnexin. Our results demonstrate that palmitoylated calnexin interacts with sarcoendoplasmic reticulum (SR) Ca 2+ transport ATPase (SERCA) 2b and that this interaction determines ER Ca 2+ content and the regulation of ER-mitochondria Ca 2+ crosstalk. In contrast, non-palmitoylated calnexin interacts with the oxidoreductase ERp57 and performs its well-known function in quality control. Interestingly, our results also show that calnexin palmitoylation is an ER-stress-dependent mechanism. Following a short-term ER stress, calnexin quickly becomes less palmitoylated, which shifts its function from the regulation of Ca 2+ signaling towards chaperoning and quality control of known substrates. These changes also correlate with a preferential distribution of calnexin to the MAM under resting conditions, or the rough ER and ER quality control compartment (ERQC) following ER stress. Our results have therefore identified the switch that assigns calnexin either to Ca 2+ signaling or to protein chaperoning.

Huntington disease (HD) is a debilitating neurodegenerative disease characterized by the loss of motor control and cognitive ability that ultimately leads to death. It is caused by the expansion of a polyglutamine tract in the huntingtin (HTT) protein, which leads to aggregation of the protein and eventually cellular death. Both the wild-type and mutant form of the protein are highly regulated by post-translational modifications including proteolysis, palmitoylation and phosphorylation. We now demonstrate the existence of a new post-translational modification of HTT: the addition of the 14 carbon fatty acid myristate to a glycine residue exposed on a caspase-3-cleaved fragment (post-translational myristoylation) and that myristoylation of this fragment is altered in a physiologically relevant model of mutant HTT. Myristoylated HTT553-585-EGFP, but not its non-myristoylated variant, initially localized to the ER, induced the formation of autophagosomes and accumulated in abnormally large autophagolysosomal/lysosomal structures in a variety of cell types, including neuronal cell lines under nutrient-rich conditions. Our results suggest that accumulation of myristoylated HTT553-586 in cells may alter the rate of production of autophagosomes and/or their clearance through the heterotypic autophagosomal/lysosomal fusion process. Overall, our novel observations establish a role for the post-translational myristoylation of a caspase-3-cleaved fragment of HTT, highly similar to the Barkor/ATG14L autophagosome-targeting sequence domain thought to sense, maintain and/or promote membrane curvature in the regulation of autophagy. Abnormal processing or production of this myristoylated HTT fragment might be involved in the pathophysiology of HD.

Myristoylation, the N-terminal modification of proteins with the fatty acid myristate, is critical for membrane targeting and cell signaling. Because cancer cells often have increased N-myristoyltransferase (NMT) expression, NMTs were proposed as anti-cancer targets. To systematically investigate this, we performed robotic cancer cell line screens and discovered a marked sensitivity of hematological cancer cell lines, including B-cell lymphomas, to the potent pan-NMT inhibitor PCLX-001. PCLX-001 treatment impacts the global myristoylation of lymphoma cell proteins and inhibits early B-cell receptor (BCR) signaling events critical for survival. In addition to abrogating myristoylation of Src family kinases, PCLX-001 also promotes their degradation and, unexpectedly, that of numerous non-myristoylated BCR effectors including c-Myc, NFκB and P-ERK, leading to cancer cell death in vitro and in xenograft models. Because some treated lymphoma patients experience relapse and die, targeting B-cell lymphomas with a NMT inhibitor potentially provides an additional much needed treatment option for lymphoma.

Myristoylation occurs cotranslationally on nascent proteins and post-translationally during apoptosis after caspase cleavages expose cryptic myristoylation sites. We demonstrate a drastic change in the myristoylated protein proteome in apoptotic cells, likely as more substrates are revealed by caspases. We show for the first time that both N-myristoyltransferases (NMTs) 1 and 2 are cleaved during apoptosis and that the caspase-3- or -8-mediated cleavage of NMT1 at Asp-72 precedes the cleavage of NMT2 by caspase-3 mainly at Asp-25. The cleavage of NMTs did not significantly affect their activity in apoptotic cells until the 8 h time point. However, the cleavage of the predominantly membrane bound NMT1 (64%) removed a polybasic domain stretch and led to a cytosolic relocalization (>55%), whereas predominantly cytosolic NMT2 (62%) relocalized to membranes when cleaved (>80%) after the removal of a negatively charged domain. The interplay between caspases and NMTs during apoptosis is of particular interest since caspases may not only control the rates of substrate production but also their myristoylation rate by regulating the location and perhaps the specificity of NMTs. Since apoptosis is often suppressed in cancer, the reduced caspase activity seen in cancer cells might also explain the higher NMT levels observed in many cancers.

Myristoylation, the addition of a 14-carbon fatty acid to the N-terminal glycine of a protein, is key to protein-membrane and protein-protein interactions. Typically, myristoylation occurs cotranslationally; however, post-translational myristoylation of caspase-cleaved proteins is now emerging as a well-established protein modification and as a novel regulator of apoptosis. To identify additional post-translationally myristoylated proteins, we engineered a plasmid vector encoding for a caspase-cleavable reporter protein named tandem reporter assay for myristoylation of proteins post-translationally (TRAMPP). pTRAMPP consists of tdTomato-DEVD-"test myristoylation sequence"-enhanced green fluorescent protein (EGFP). After induction of apoptosis, the reporter protein is cleaved by caspases, which frees a new N-terminal glycine residue attached to EGFP that can be myristoylated. We used pTRAMPP in appropriately transfected cells to identify 7 post-translationally myristoylated proteins. First, we confirmed the post-translational myristoylation of two previously identified putative substrates, cytoplasmic dynein intermediate chain 2A and PKCε (ctPKCε), and identified 5 more caspase-cleaved potential substrates for myristoylation that include the antiapoptotic regulator of apoptosis, Mcl-1, and the causative agent of Huntington's disease, huntingtin protein. Further investigation revealed that post-translationally myristoylated ctPKCε localized to membranes and increased Erk signaling and degradation of the proapoptotic protein Bim, which prevented a significant loss of mitochondrial potential of 17% over nonmyristoylated ctPKCε in HeLa cells in the presence of apoptotic stimuli. Taken together, these findings suggest a possible antiapoptotic role for post-translationally myristoylated caspase-cleaved ctPKCε.

Chaperones in the endoplasmic reticulum (ER) control the flux of Ca2+ ions into mitochondria, thereby increasing or decreasing the energetic output of the oxidative phosphorylation pathway. An example is the abundant ER lectin calnexin, which interacts with sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). We found that calnexin stimulated the ATPase activity of SERCA by maintaining its redox state. This function enabled calnexin to control how much ER Ca2+ was available for mitochondria, a key determinant for mitochondrial bioenergetics. Calnexin-deficient cells compensated for the loss of this function by partially shifting energy generation to the glycolytic pathway. These cells also showed closer apposition between the ER and mitochondria. Calnexin therefore controls the cellular energy balance between oxidative phosphorylation and glycolysis.