Identification of Secret Agent as the O- GlcNAc Transferase That Participates in Plum Pox Virus Infection

Abstract

Serine and threonine of many nuclear and cytoplasmic proteins are posttranslationally modified with O-linked N-acetylglucosamine (O-GlcNAc). This modification is made by O-linked N-acetylglucosamine transferases (OGTs). Genetic and biochemical data have demonstrated the existence of two OGTs of Arabidopsis thaliana, SECRET AGENT (SEC) and SPINDLY (SPY), with at least partly overlapping functions, but there is little information on their target proteins. The N terminus of the capsid protein (CP) of Plum pox virus (PPV) isolated from Nicotiana clevelandii is O-GlcNAc modified. We show here that O-GlcNAc modification of PPV CP also takes place in other plant hosts, N. benthamiana and Arabidopsis. PPV was able to infect the Arabidopsis OGT mutants sec-1, sec-2, and spy-3, but at early times of the infection, both rate of virus spread and accumulation were reduced in sec-1 and sec-2 relative to spy-3 and wild-type plants. By matrix-assisted laser desorption ionization-time of flight mass spectrometry, we determined that a 39-residue tryptic peptide from the N terminus of CP of PPV purified from the spy-3 mutant, but not sec-1 or sec-2, was O-GlcNAc modified, suggesting that SEC but not SPY modifies the capsid. While our results indicate that O-GlcNAc modification of PPV CP by SEC is not essential for infection, they show that the modification has a role(s) in the process.Dynamic modification of serine and threonine with O-linked ␤-N-acetylglucosamine (O-GlcNAcylation) is widespread among nuclear and cytoplasmic eukaryotic proteins (33). The modification is essential for viability in both plants and animals (9, 23). O-GlcNAcylation is a regulatory modification that shares many common traits with protein phosphorylation (26). In addition, it has been documented for several proteins that phosphorylation and O-GlcNAcylation can occur at the same site. While the details of how O-GlcNAcylation functions in specific pathways remain to be elucidated, some general themes have emerged. The level of O-GlcNAcylation is dynamic and is influenced by hormonal signals, stress, and metabolic status, and the modification has roles in the regulation of transcription and protein synthesis and degradation. Disturbance of cellular processes regulated by O-GlcNAcylation appears to be involved in very important pathological disorders such as Alzheimer's disease (17) and diabetes (31).The enzymes responsible for O-GlcNAc addition (O-GlcNAc transferases [OGTs]) are highly conserved in plants and animals (33). OGT enzymes have an N-terminal tetratricopeptide repeat domain and a C-terminal catalytic domain. While animals have one OGT, Arabidopsis thaliana has two: Secret Agent (SEC) and SPINDLY (SPY) (9, 29). Genetic experiments have demonstrated that SEC and SPY have at least partly overlapping functions (9), but so far, there is very little information on their target proteins.O-GlcNAc modifications have been found in some structural proteins from different animal viruses such as the cytomegalovirus basic phosphoprotein (8), the adenovirus fi...

A Novel Role of the Potyviral Helper Component Proteinase Contributes To Enhance the Yield of Viral Particles

“…On one hand, CP is phosphorylated by the protein kinase CK2, a modification that seems to regulate the RNA binding function of CP and therefore likely controls the formation and/or stability of viral ribonucleoproteins (59)(60)(61). On the other hand, O-GlcNAcylation modifications carried out by the host protein SECRET AGENT were also found in PPV CP (59,62). Interestingly, a mutagenic approach showed that O-GlcNAcylation might have a role in virion assembly and/or stability (63).…”

Unfolding the secrets of plum pox virus: from epidemiology to genomics

“…This fact is inconsistent with variable CP mobility in SDS-PAGE, which is apparently caused by non-amino acid compounds coupled with CP molecules by posttranslational modification (Šubr and Glasa, 2004;Kollerová et al, 2008). O-glycosylation of CP serine and threonine residues by N-acetylglycosamine has been detected and the corresponding plant enzyme was identified (Fernández-Fernández et al, 2002;Chen et al, 2005;Scott et al, 2006). Localization of this modification in the N-terminal CP part is in agreement with the fact that core CP of different PPV strains migrated equally in SDS-PAGE Šubr and Glasa, 1999).…”

Functional analysis of HvSPY, a negative regulator of GA response, in barley aleurone cells and Arabidopsis

“…Two OGT proteins have been identiWed in Arabidopsis-SPY and SECRET AGENT (SEC)-the latter being more closely related to human OGT (Hartweck et al 2002). It has been demonstrated that SEC is involved in the O-GlcNAc modiWcation of the Nterminus of a capsid protein of the plum pox virus (Chen et al 2005). However, to date, no proteins modiWed by SPY have been reported.…”