Localization of the Palmitoylation Site in the Transmembrane Protein p12E of Friend Murine Leukaemia Virus

Abstract

Friend murine leukaemia virus complex was propagated on murine cells in the presence of 19,IO-'Hlpalmitic acid. Virus particles were harvested from the culture supernatant and lysed with detergents. The viral transmembrane protein, pl2E, was isolated from the lysates by size-exclusion chromatography and purified by narrowbore reverse-phase HPLC. Analysis of the purified product by matrix-assisted laser desorptiodionization time-of-flight mass spectrometry (MALDT-TOF-MS) revealed that the protein is palmitoylated carrying one fatty acid residue. The radiolabelled fatty acid was released by hydroxylamine treatment at pH 7, indicating that acylation occurred via a thioester linkage. For allocation of the acylation site, p12E was digested with trypsin. The resulting peptides were either directly subjected to MALDI-TOF-MS or fractionated by microbore reverse-phase HPLC prior to mass spectrometry. The results revealed that p12E of Friend murine leukaemia virus is acylated at a cysteine residue situated at the Cterminal side of the putative transmembrane anchor of the polypeptide. Fatty acid analysis of the purified acylpeptide demonstrated that p12E carries almost exclusively palmitic acid.Keywords: fatty acid; mass spectrometry ; protein acylation ; retrovirus.The envelope (env) gene products of Friend murine leukaemia virus (F-MuLV) are synthesized as a common precursor molecule, gPr90, that is proteolytically cleaved into the viral glycoprotein, gp71, and the nonglycosylated transmembrane protein, p15E [I]. In a second step, p15E is processed by a virusencoded protease yielding p12E [2, 31. The retroviral envelope glycoprotein mediates the first contact with specific host cell receptors [4-81 thus determining the viral host range 18, 91. The corresponding transmembrane protein, p12E, functions as an anchor for gp71. Furthermore, it is substantially involved in the fusion of viral and cellular membranes [lo, 111. It has been demonstrated that the membrane fusion capability is greatly enhanced by processing of p15E to p12E [12]. Hence, both env gene products play an important role in viral pathogenesis [13].Using metabolic labelling with ['Hlpalmitic acid, evidence has been provided that F-MuLV p12E might be post-translationally acylated [14]. However, there is no direct proof that a fatty acid is covalently linked to p12E. Furthermore, the fatty acid substitution pattern, the type of linkage, and the acylation site have not been investigated. Since palmitoylation of proteins has been reported to play an important role in controlling their intracellular location and biological functions [15 -181, we have analysed the acylation of F-MuLV p12E in detail. The results obCorrespondence to R. Geyer, Biochemisches Institut am Klinikum Fax: +49 641 702 4103. Abbreviations. CF,CO,H, trifluoroacetic acid ; F-MuLV, Friend murine leukaemia virus; gp71, 71-kDa glycoprotein; gPr90, primary translation product of the envelope gene; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry ; p12E and...

Palmitoyl acyltransferase assays and inhibitors (Review)

“…For the analysis of palmitoylated proteins, the lipid moiety is removed by treatment with hydroxylamine, which shifts the mass of the depalmitoylated peptide. Comparing the peptide masses with or without hydroxylamine treatment, allows the site of palmitoylation to be inferred [21]. An advantage of using MS is that it allows the estimation of the stoichiometry of the palmitoylation of a particular protein of interest.…”

Palmitoylation of the feline immunodeficiency virus envelope glycoprotein and its effect on fusion activity and envelope incorporation into virions

“…Several retroviral glycoproteins have been found to be palmitoylated. In both Friend and Moloney murine leukemia viruses (F-MuLV and M-MuLV, respectively) palmitoylation occurs at the cysteine residue within the TM membrane-spanning domain (Hensel et al, 1995;Yang and Compans, 1996). In M-MuLV substitution of this cysteine with serine reduces Env association with lipid rafts, although it should be taken into account that this mutation also impairs Env transport to the cell surface (Li et al, 2002).…”

Cholesterol Depletion Inactivates XMRV and Leads to Viral Envelope Protein Release from Virions: Evidence for Role of Cholesterol in XMRV Infection

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