Regulation of enzymatic activity by active site fatty acylation. A new role for long chain fatty acid acylation of proteins.

Methylmalonate semialdehyde dehydrogenase (MMSDH) is a mitochondrial enzyme which can be acylated by myristoyl-CoA analogs (Deichaite, I., Berthiaume, L., Peseckis, S. M., Patton, W. F., and Resh, M. D. (1993) J. Biol. Chem. 268, 13788-13747). Here we describe the mechanisms which mediate regulation of the enzymatic activity of bovine MMSDH by long chain fatty acylation. The substrate specificity of the acylation reaction was measured in vitro using purified MMSDH and the coenzyme A derivative of an 125I-labeled long chain fatty acid (13-iodotridecanoate), an analog of myristoyl-CoA. Long chain fatty acyl CoAs (> 8 carbons) were able to inhibit radiolabeling of MMSDH. In order to study the physiological role of the acylation process in vivo, a system using highly purified mitochondria from COS-1 cells overexpressing MMSDH was exploited. MMSDH was shown to be processed properly, targeted to the mitochondrial fraction, and enzymatically active. The extent of fatty acylation of MMSDH as well as of other mitochondrial proteins was correlated with the mitochondrial energy level. Biochemical evidence as well as site-specific mutagenesis of cysteine 319 revealed that this highly conserved active site cysteine of MMSDH was the target of the fatty acylation. Another member of the aldehyde dehydrogenase family, yeast aldehyde dehydrogenase was also covalently modified by [125I]13-iodotridecanoyl-CoA and thereby inactivated. Furthermore, we demonstrate that glutamate dehydrogenase, an enzyme that has been previously shown to be strongly inhibited by palmitoyl-CoA, is fatty acylated by the 125I-labeled myristoyl-CoA analog. Our data suggest that attachment of long chain fatty acids to proteins is a new and potentially widespread type of enzyme regulation mechanism that we denote active site fatty acylation.

Novel use of an iodo-myristyl-CoA analog identifies a semialdehyde dehydrogenase in bovine liver.

We describe here the identification, purification, and characterization of a semialdehyde dehydrogenase with a novel fatty acid binding function. The coenzyme A derivative of an 125I-labeled long chain saturated fatty acid (13-iodo-tridecanoate) was used to tag proteins which bind myristoyl-CoA. A prominent 57 kDa band was identified, which was isolated from bovine liver by a high salt extraction followed by ammonium sulfate precipitation. Sequential chromatographic separation using phenyl-Sepharose, hydroxyapatite, DEAE-Sepharose, Mono Q, and Fast Flow S resins resulted in a purified protein that migrated as a single band of 57 kDa on denaturing gels. Sephacryl-200 gel filtration provided a native molecular mass estimation of 118 kDa suggesting that this protein exists as a dimer. Two-dimensional gel analysis resolved three isoform variants with pI values of 7.4, 7.7, and 7.9, respectively, and established that the pI = 7.9 form has the highest propensity for fatty acid binding. We proceeded to generate tryptic peptides from the purified protein and subjected several peptides to microchemical sequencing. Degenerate oligonucleotide probes were designed and polymerase chain reaction was used to generate a unique nucleotide sequence. Subsequent screening of a bovine liver cDNA library yielded a 1.7-kilobase clone which encodes a protein of 537 amino acids (58 kDa) with 95% identity to mammalian methylmalonate semialdehyde dehydrogenase (MMSDH). In vitro assays confirmed that the purified 57-kDa protein exhibited MMSDH activity, and that preincubation of the enzyme with fatty acyl-CoA inhibited its dehydrogenase activity. The myristyl-CoA analog therefore serves as an affinity label for MMSDH. We propose that fatty acyl CoAs may have the potential to function as enzyme regulators in vivo.

Iodinated fatty acids as probes for myristate processing and function. Incorporation into pp60v-src.

The myristyl group makes a critical contribution to the processing, trafficking, and function of myristylated proteins. A series of [omega-125I]iodo-fatty acids was synthesized in order to elucidate the myristyl group's contribution to the membrane association of pp60v-src, the transforming protein of Rous sarcoma virus. In vitro translation of v-src mRNA was employed to monitor incorporation of myristyl analogs into pp60v-src polypeptide. 12-Iodododecanoic, 13-iodotridecanoic, and 14-iodotetradecanoic acids were selectively incorporated in vitro into pp60v-src. One-dimensional peptide analysis confirmed that the analogs were attached to the N terminus of pp60v-src. Upon addition of membranes, the Src proteins modified by these analogs bound to membranes at levels comparable with or slightly less than the myristyl parent. Myristyl analogs were also shown to be incorporated into pp60v-src in vivo. Fractionation of [omega-125I]iodo-fatty acid labeled cells showed that 12-iododecanoic acid, 13-iodotridecanoic acid, and 14-iodotetradecanoic acid modified pp60v-src associated preferentially with the membrane fractions. These results demonstrate that fatty acyl groups one carbon longer or shorter than myristate can be accommodated within the membrane binding site for pp60v-src and illustrate the utility of in vitro systems for predicting analog behavior in vivo. We anticipate that iodinated fatty acids can be used as tools to aid in clarifying the role of the fatty acid in a variety of myristylated molecules.

In vitro synthesis of pp60v-src: myristylation in a cell-free system.

Covalent attachment of myristic acid to pp60v-src, the transforming protein of Rous sarcoma virus, was studied in a cell-free system. Using a synthetic peptide containing the first 11 amino acids of the mature pp60v-src polypeptide sequence as a substrate, we probed lysates from a variety of cells and tissues for N-myristyl transferase (NMT) activity. Nearly every eucaryotic cell type tested contained NMT, including avian, mammalian, insect, and plant cells. Since NMT activity was detected in rabbit reticulocyte lysates, we took advantage of the translational capability of these lysates to determine the precise point during translation at which myristate is attached to pp60v-src. src mRNA, transcribed from cloned v-src DNA, was translated in reticulocyte lysates which had been depleted of endogenous myristate. Addition of [3H]myristate to lysates 10 min after the start of synchronized translation resulted in a dramatic decrease in the incorporation of radiolabeled myristate into pp60v-src polypeptide chains. These results imply that although myristate can be attached posttranslationally to synthetic peptide substrates, myristylation in vivo is apparently a very early cotranslational event which occurs before the first 100 amino acids of the nascent polypeptide chain are polymerized.

Linear simian virus 40 DNA fragments exhibit a propensity for rolling-circle replication.

A linear simian virus 40 origin-containing DNA fragment replicated in monkey COS cells, generating tandemly repeated (head-to-tail) structures. Electron microscopy revealed circle-and-tail configurations characteristic of rolling-circle replication intermediates. Circularization of the same DNA before transfection led to a theta type of replication which generated supercoiled DNA molecules.

Circular and linear simian virus 40 DNAs differ in recombination.

Linear forms of simian virus 40 (SV40) DNA, when added to transfection mixtures containing circular SV40 and phi X174 RFI DNAs, enhanced the frequency of SV40/phi X174 recombination, as measured by infectious center in situ plaque hybridization in monkey BSC-1 cells. The sequences required for the enhancement of recombination by linear DNA reside within the SV40 replication origin/regulatory region (nucleotides 5,171 to 5,243/0 to 128). Linearization of phi X174 RFI DNA did not increase the recombination frequency. The SV40/phi X174 recombinant structures arising from transfections supplemented with linear forms of origin-containing SV40 DNA contained phi X174 DNA sequences interspersed within tandem head-to-tail repeats derived from the recombination-enhancing linear DNA. Evidence is presented that the tandem repeats are not formed by homologous recombination and that linear forms of SV40 DNA must compete with circular SV40 DNA for the available T antigen to enhance recombination. We propose that the enhancement of recombination by linear SV40 DNA results from the entry of that DNA into a rolling circle type of replication pathway which generates highly recombinogenic intermediates.