Blocked O-GlcNAc cycling alters mitochondrial morphology, function, and mass

Elizabeth O Akinbiyi,Maria S K Stoll,Chao-Pin Hsiao,Brianna L Bauer,John A Hanover,Jason A Mears,Charles L Hoppel,Lara K Abramowitz

doi:10.1038/s41598-021-01512-y

Abstract

O-GlcNAcylation is a prevalent form of glycosylation that regulates proteins within the cytosol, nucleus, and mitochondria. The O-GlcNAc modification can affect protein cellular localization, function, and signaling interactions. The specific impact of O-GlcNAcylation on mitochondrial morphology and function has been elusive. In this manuscript, the role of O-GlcNAcylation on mitochondrial fission, oxidative phosphorylation (Oxphos), and the activity of electron transport chain (ETC) complexes were evaluated. In a cellular environment with hyper O-GlcNAcylation due to the deletion of O-GlcNAcase (OGA), mitochondria showed a dramatic reduction in size and a corresponding increase in number and total mitochondrial mass. Because of the increased mitochondrial content, OGA knockout cells exhibited comparable coupled mitochondrial Oxphos and ATP levels when compared to WT cells. However, we observed reduced protein levels for complex I and II when comparing normalized mitochondrial content and reduced linked activity for complexes I and III when examining individual ETC complex activities. In assessing mitochondrial fission, we observed increased amounts of O-GlcNAcylated dynamin-related protein 1 (Drp1) in cells genetically null for OGA and in glioblastoma cells. Individual regions of Drp1 were evaluated for O-GlcNAc modifications, and we found that this post-translational modification (PTM) was not limited to the previously characterized residues in the variable domain (VD). Additional modification sites are predicted in the GTPase domain, which may influence enzyme activity. Collectively, these results highlight the impact of O-GlcNAcylation on mitochondrial dynamics and ETC function and mimic the changes that may occur during glucose toxicity from hyperglycemia.

Highlights

O-GlcNAcylation is a prevalent form of glycosylation that regulates proteins within the cytosol, nucleus, and mitochondria
Our results demonstrate the impact of a genetic deletion induced global hyper-O-GlcNAcylation cellular environment on Drp[1] O-GlcNAc modifications, changes in mitochondrial morphology, mitochondrial oxidative phosphorylation (Oxphos) and activity of electron transport chain (ETC) complexes not seen in previous studies using pharmacologic inhibitors
We found that mitochondrial morphology was predominantly intermediate and tubular in the basal WT mouse embryonic fibroblasts (MEFs), making up 51% and 39% of measured mitochondria in these cells respectively (Fig. 1A,C,E)

Summary

Introduction

O-GlcNAcylation is a prevalent form of glycosylation that regulates proteins within the cytosol, nucleus, and mitochondria. Drp[1] is an 80 kDa cytosolic protein that is actively recruited to the surface of mitochondria to promote membrane remodeling This process is tightly regulated through lipid and protein interactions as well as a diverse collection of post-translational modifications, including phosphorylation, nitrosylation, ubiquitylation, sumoylation, and O-GlcNAcylation[12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]. While organelle elongation is proposed to enhance electron transport chain (ETC) activity, increased fission limits oxidative phosphorylation (Oxphos)[33] This may be due to changes in cristae morphology or other differences in membrane architecture that alter assembly of individual ETC complexes and larger respiratory s upercomplexes[34,35]. The indirect consequences of small molecules that alter PTM signaling and the diversity of assays used to assess changes in mitochondrial function have complicated interpretations

Methods

Results

Conclusion