N-glycosylation of Haloferax volcanii flagellins requires known Agl proteins and is essential for biosynthesis of stable flagella.
N-glycosylation, a posttranslational modification required for the accurate folding and stability of many proteins, has been observed in organisms of all domains of life. Although the haloarchaeal S-layer glycoprotein was the first prokaryotic glycoprotein identified, little is known about the glycosylation of other haloarchaeal proteins. We demonstrate here that the glycosylation of Haloferax volcanii flagellins requires archaeal glycosylation (Agl) components involved in S-layer glycosylation and that the deletion of any Hfx. volcanii agl gene impairs its swimming motility to various extents. A comparison of proteins in CsCl density gradient centrifugation fractions from supernatants of wild-type Hfx. volcanii and deletion mutants lacking the oligosaccharyltransferase AglB suggests that when the Agl glycosylation pathway is disrupted, cells lack stable flagella, which purification studies indicate consist of a major flagellin, FlgA1, and a minor flagellin, FlgA2. Mass spectrometric analyses of FlgA1 confirm that its three predicted N-glycosylation sites are modified with covalently linked pentasaccharides having the same mass as that modifying its S-layer glycoprotein. Finally, the replacement of any of three predicted N-glycosylated asparagines of FlgA1 renders cells nonmotile, providing direct evidence for the first time that the N-glycosylation of archaeal flagellins is critical for motility. These results provide insight into the role that glycosylation plays in the assembly and function of Hfx. volcanii flagella and demonstrate that Hfx. volcanii flagellins are excellent reporter proteins for the study of haloarchaeal glycosylation processes.
Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Manuela
M
Tripepi
Jason
J
You
Sevcan
S
Temel
Özlem
Ö
Önder
Dustin
D
Brisson
Mechthild
M
Pohlschröder
D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D005407:Flagella / Q000378:metabolism*; D005408:Flagellin / Q000378:metabolism*; D017353:Gene Deletion; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D019622:Haloferax volcanii / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D008124:Locomotion; D008954:Models, Biological; D011499:Protein Processing, Post-Translational
D019843:Archaeal Proteins; D005408:Flagellin; D016695:Glycosyltransferases