Quantitative glycoproteomics identifies target N-glycoproteins of the Golgi-class I α-mannosidase SlMNSI1 that is important for tomato fruit development.

Abstract

Protein N-glycosylation is a fundamental post-translational modification, yet its role in regulating fleshy fruit development is poorly understood. Here we combined chemical genetics, CRISPR/Cas9-mediated gene editing, quantitative site-specific glycoproteomics, and genetic complementation analyses to investigate the function of the class I α-mannosidase SlMNSI1 in tomato (Solanum lycopersicum). Kifunensine (Kif) inhibition or knockout of SlMNSI1 caused severe pleiotropic defects, including impaired fruit development, and led to the accumulation of Man₉GlcNAc₂ and Man₈GlcNAc₂ respectively on its expected glycoprotein substrates. To identify its targets, we generated the most comprehensive site-specific N-glycoproteome of tomato fruit to date and identified 3,091 intact N-glycopeptides containing 873 N-glycosites and 158 N-glycans within 573 N-glycoproteins. By comparing the Kif-injected tomatoes against Mock and slmnsl1 against WT, 97 N-glycoproteins with 127 high-confidence N-glycosites were identified as candidate target N-glycoproteins of SlMNSI1. Strikingly, we discovered that the Golgi-localized SlMNSI1 is itself an N-glycoprotein. Mutagenesis of N-glycosites demonstrated that N-glycosylation at Asn288 and Asn334 in SlMNSI1 is essential for its proper Golgi localization and protein stability. Genetic complementation assays confirmed this in vivo: while overexpression of the wild-type SlMNSI1 rescued the fruit development defects of the mutant, overexpression of a non-glycosylatable version of SlMNSI1 only partially complemented these phenotypes. Collectively, our findings reveal the critical role of SlMNSI1 and the processing of mannosidic N-glycans in fruit development. Identifying the SlMNSI1 N-glycoprotein substrates could help to further investigate the role of N-glycans in this process.