Lommel M, Schott A, Jank T, Hofmann V, Strahl S

Protein O-mannosylation is an essential modification among fungi and mammals. It is initiated at the endoplasmic reticulum (ER) by a conserved family of dolichyl phosphate-mannose:protein O-mannosyltransferases (PMTs). PMTs are integral membrane proteins with two hydrophilic loops (loop1 and loop5) facing the ER lumen. Formation of dimeric PMT complexes is crucial ...

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for mannosyltransferase activity, but the direct cause is not known to date. In baker(')s yeast, O-mannosylation is largely catalyzed by heterodimeric Pmt1p/Pmt2p and homodimeric Pmt4p complexes. To further characterize Pmt1p/Pmt2p complexes we developed a photoaffinity probe based on the artificial mannosyl acceptor substrate Tyr-Ala-Thr-Ala-Val. The photoreactive probe was preferentially crosslinked to Pmt1p, and deletion of the loop1 but not the loop5 region abolished this interaction. Analysis of Pmt1p loop1 mutants revealed that especially Glu-78 is crucial for binding of the photoreactive probe. Glu-78 belongs to a Asp-Glu motif that is highly conserved among PMTs. We further demonstrate that single amino acid substitutions in this motif completely abolish activity of Pmt4p complexes. In contrast, both acidic residues need to be exchanged to eliminate activity of Pmt1p/Pmt2p complexes. Based on our data, we propose that the loop1 regions of dimeric complexes form part of the catalytic site.

Date: Sep. 28, 2011

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