Quantitative glycoproteomics identifies target N-glycoproteins of the Golgi-class I α-mannosidase SlMNSI1 that is...

Protein N-glycosylation is a fundamental post-translational modification, yet its role in regulating fruit development remains poorly understood. Here, we investigated the function of the class I α-mannosidase SlMNSI1 in tomato (Solanum lycopersicum) using chemical genetics, CRISPR-Cas9-mediated gene editing, quantitative site-specific glycoproteomics, and genetic complementation analyses. Inhibition of SlMNSI by kifunensine or SlMNSI1 knockout caused severe pleiotropic defects and impaired fruit development, leading to the accumulation of Man₉GlcNAc₂ or Man₈GlcNAc₂, respectively, on glycoprotein substrates. Analysis of the tomato fruit site-specific N-glycoproteome identified 3091 intact N-glycopeptides containing 873 N-glycosites and 158 N-glycans within 573 N-glycoproteins. Among these, 97 N-glycoproteins containing 127 N-glycosites were identified as candidate targets of SlMNSI1. Strikingly, SlMNSI1 itself is N-glycosylated at Asn288 and Asn334, and N-glycosylation is essential for Golgi localization and protein stability. Genetic complementation assays verified this in vivo: overexpression of wild-type SlMNSI1 fully restored normal fruit development in the mutant, whereas overexpression of a non-glycosylatable SlMNSI1 variant only partially complemented these phenotypes. Collectively, our findings reveal the critical role of SlMNSI1 and mannosidic N-glycans in fruit development.