Dataset Information

Structure of the eukaryotic protein O-mannosyltransferase Pmt1-Pmt2 complex.

ABSTRACT: In eukaryotes, a nascent peptide entering the endoplasmic reticulum (ER) is scanned by two Sec61 translocon-associated large membrane machines for protein N-glycosylation and protein O-mannosylation, respectively. While the structure of the eight-protein oligosaccharyltransferase complex has been determined recently, the structures of mannosyltransferases of the PMT family, which are an integral part of ER protein homeostasis, are still unknown. Here we report cryo-EM structures of the Saccharomyces cerevisiae Pmt1-Pmt2 complex bound to a donor and an acceptor peptide at 3.2-Å resolution, showing that each subunit contains 11 transmembrane helices and a lumenal ?-trefoil fold termed the MIR domain. The structures reveal the substrate recognition model and confirm an inverting mannosyl-transferring reaction mechanism by the enzyme complex. Furthermore, we found that the transmembrane domains of Pmt1 and Pmt2 share a structural fold with the catalytic subunits of oligosaccharyltransferases, confirming a previously proposed evolutionary relationship between protein O-mannosylation and protein N-glycosylation.

SUBMITTER: Bai L

PROVIDER: S-EPMC6684406 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Structure of the eukaryotic protein O-mannosyltransferase Pmt1-Pmt2 complex.

Bai Lin L Kovach Amanda A You Qinglong Q Kenny Alanna A Li Huilin H

Nature structural & molecular biology 20190708 8

In eukaryotes, a nascent peptide entering the endoplasmic reticulum (ER) is scanned by two Sec61 translocon-associated large membrane machines for protein N-glycosylation and protein O-mannosylation, respectively. While the structure of the eight-protein oligosaccharyltransferase complex has been determined recently, the structures of mannosyltransferases of the PMT family, which are an integral part of ER protein homeostasis, are still unknown. Here we report cryo-EM structures of the Saccharom ...[more]

PMID: 31285605

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Project description:The family of mammalian O-mannosyltransferases includes two enzymes, POMT1 and POMT2, which are thought to be essential for muscle and neural development. Similar to mammalian organisms, Drosophila has two O-mannosyltransferase genes, rotated abdomen (rt) and DmPOMT2, encoding proteins with high homology to their mammalian counterparts. The previously reported mutant phenotype of the rt gene includes a clockwise rotation of the abdomen and defects in embryonic muscle development. No mutants have been described so far for the DmPOMT2 locus. In this study, we determined that the mutation in the twisted (tw) locus, tw(1), corresponds to a DmPOMT2 mutant. The twisted alleles represent a complementation group of recessive mutations that, similar to the rt mutants, exhibit a clockwise abdomen rotation phenotype. Several tw alleles were isolated in the past; however, none of them was molecularly characterized. We used an expression rescue approach to confirm that tw locus represents DmPOMT2 gene. We found that the tw1 allele represents an amino acid substitution within the conserved PMT domain of DmPOMT2 (TW) protein. Immunostaining experiments revealed that the protein products of both rt and tw genes colocalize within Drosophila cells where they reside in the ER subcellular compartment. In situ hybridization analysis showed that both genes have essentially overlapping patterns of expression throughout most of embryogenesis (stages 8-17), while only the rt transcript is present at early embryonic stages (5 and 6), suggesting its maternal origin. Finally, we analyzed the genetic interactions between rt and tw using several mutant alleles, RNAi, and ectopic expression approaches. Our data suggest that the two Drosophila O-mannosyltransferase genes, rt and tw, have nonredundant functions within the same developmental cascade and that their activities are required simultaneously for possibly the same biochemical process. Our results establish the possibility of using Drosophila as a model system for studying molecular and genetic mechanisms of protein O-mannosylation during development.

Dataset Information

Structure of the eukaryotic protein O-mannosyltransferase Pmt1-Pmt2 complex.

Publications

Structure of the eukaryotic protein O-mannosyltransferase Pmt1-Pmt2 complex.

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