The tobacco GNTI stem region harbors a strong motif for homomeric protein complex formation
IntroductionThe Golgi apparatus of plants is the central cellular organelle for glycan processing and polysaccharide biosynthesis. These essential processes are catalyzed by a large number of Golgi-resident glycosyltransferases and glycosidases whose organization within the Golgi is still poorly und...
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| Format: | Article |
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Frontiers Media S.A.
2023-11-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2023.1320051/full |
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| author | Jennifer Schoberer Shiva Izadi Carolina Kierein Ulrike Vavra Julia König-Beihammer Valentina Ruocco Clemens Grünwald-Gruber Alexandra Castilho Richard Strasser |
| author_facet | Jennifer Schoberer Shiva Izadi Carolina Kierein Ulrike Vavra Julia König-Beihammer Valentina Ruocco Clemens Grünwald-Gruber Alexandra Castilho Richard Strasser |
| author_sort | Jennifer Schoberer |
| collection | DOAJ |
| description | IntroductionThe Golgi apparatus of plants is the central cellular organelle for glycan processing and polysaccharide biosynthesis. These essential processes are catalyzed by a large number of Golgi-resident glycosyltransferases and glycosidases whose organization within the Golgi is still poorly understood.MethodsHere, we examined the role of the stem region of the cis/medial Golgi enzyme N-acetylglucosaminyltransferase I (GNTI) in homomeric complex formation in the Golgi of Nicotiana benthamiana using biochemical approaches and confocal microscopy.ResultsTransient expression of the N-terminal cytoplasmic, transmembrane, and stem (CTS) regions of GNTI leads to a block in N-glycan processing on a co-expressed recombinant glycoprotein. Overexpression of the CTS region from Golgi α-mannosidase I, which can form in planta complexes with GNTI, results in a similar block in N-glycan processing, while GNTI with altered subcellular localization or N-glycan processing enzymes located further downstream in the Golgi did not affect complex N-glycan processing. The GNTI-CTS-dependent alteration in N-glycan processing is caused by a specific nine-amino acid sequence motif in the stem that is required for efficient GNTI-GNTI interaction.DiscussionTaken together, we have identified a conserved motif in the stem region of the key N-glycan processing enzyme GNTI. We propose that the identified sequence motif in the GNTI stem region acts as a dominant negative motif that can be used in transient glycoengineering approaches to produce recombinant glycoproteins with predominantly mannosidic N-glycans. |
| first_indexed | 2024-03-09T14:23:09Z |
| format | Article |
| id | doaj.art-d51270cf601b46ce9dacb6de0756f601 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-03-09T14:23:09Z |
| publishDate | 2023-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-d51270cf601b46ce9dacb6de0756f6012023-11-28T09:54:40ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2023-11-011410.3389/fpls.2023.13200511320051The tobacco GNTI stem region harbors a strong motif for homomeric protein complex formationJennifer Schoberer0Shiva Izadi1Carolina Kierein2Ulrike Vavra3Julia König-Beihammer4Valentina Ruocco5Clemens Grünwald-Gruber6Alexandra Castilho7Richard Strasser8Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, AustriaDepartment of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, AustriaDepartment of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, AustriaDepartment of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, AustriaDepartment of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, AustriaDepartment of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, AustriaCore Facility Mass Spectrometry, University of Natural Resources and Life Sciences, Vienna, AustriaDepartment of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, AustriaDepartment of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, AustriaIntroductionThe Golgi apparatus of plants is the central cellular organelle for glycan processing and polysaccharide biosynthesis. These essential processes are catalyzed by a large number of Golgi-resident glycosyltransferases and glycosidases whose organization within the Golgi is still poorly understood.MethodsHere, we examined the role of the stem region of the cis/medial Golgi enzyme N-acetylglucosaminyltransferase I (GNTI) in homomeric complex formation in the Golgi of Nicotiana benthamiana using biochemical approaches and confocal microscopy.ResultsTransient expression of the N-terminal cytoplasmic, transmembrane, and stem (CTS) regions of GNTI leads to a block in N-glycan processing on a co-expressed recombinant glycoprotein. Overexpression of the CTS region from Golgi α-mannosidase I, which can form in planta complexes with GNTI, results in a similar block in N-glycan processing, while GNTI with altered subcellular localization or N-glycan processing enzymes located further downstream in the Golgi did not affect complex N-glycan processing. The GNTI-CTS-dependent alteration in N-glycan processing is caused by a specific nine-amino acid sequence motif in the stem that is required for efficient GNTI-GNTI interaction.DiscussionTaken together, we have identified a conserved motif in the stem region of the key N-glycan processing enzyme GNTI. We propose that the identified sequence motif in the GNTI stem region acts as a dominant negative motif that can be used in transient glycoengineering approaches to produce recombinant glycoproteins with predominantly mannosidic N-glycans.https://www.frontiersin.org/articles/10.3389/fpls.2023.1320051/fullcell biologyglycoengineeringglycosylationGolgi apparatusprotein-protein interactionrecombinant protein |
| spellingShingle | Jennifer Schoberer Shiva Izadi Carolina Kierein Ulrike Vavra Julia König-Beihammer Valentina Ruocco Clemens Grünwald-Gruber Alexandra Castilho Richard Strasser The tobacco GNTI stem region harbors a strong motif for homomeric protein complex formation Frontiers in Plant Science cell biology glycoengineering glycosylation Golgi apparatus protein-protein interaction recombinant protein |
| title | The tobacco GNTI stem region harbors a strong motif for homomeric protein complex formation |
| title_full | The tobacco GNTI stem region harbors a strong motif for homomeric protein complex formation |
| title_fullStr | The tobacco GNTI stem region harbors a strong motif for homomeric protein complex formation |
| title_full_unstemmed | The tobacco GNTI stem region harbors a strong motif for homomeric protein complex formation |
| title_short | The tobacco GNTI stem region harbors a strong motif for homomeric protein complex formation |
| title_sort | tobacco gnti stem region harbors a strong motif for homomeric protein complex formation |
| topic | cell biology glycoengineering glycosylation Golgi apparatus protein-protein interaction recombinant protein |
| url | https://www.frontiersin.org/articles/10.3389/fpls.2023.1320051/full |
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