The Golgi apparatus is the central hub for protein trafficking and glycosylation in the secretory pathway. However, how the Golgi responds to glucose deprivation is so far unknown. Here, we report that GRASP55, the Golgi stacking protein located in medial- and trans-Golgi cisternae, is O-GlcNAcylated by the O-GlcNAc transferase OGT under growth conditions. Glucose deprivation reduces GRASP55 O-GlcNAcylation. De-O-GlcNAcylated GRASP55 forms puncta outside of the Golgi area, which co-localize with autophagosomes and late endosomes/lysosomes. GRASP55 depletion reduces autophagic flux and results in autophagosome accumulation, while expression of an O-GlcNAcylation-deficient mutant of GRASP55 accelerates autophagic flux. Biochemically, GRASP55 interacts with LC3-II on the autophagosomes and LAMP2 on late endosomes/lysosomes and functions as a bridge between LC3-II and LAMP2 for autophagosome and lysosome fusion; this function is negatively regulated by GRASP55 O-GlcNAcylation. Therefore, GRASP55 senses glucose levels through O-GlcNAcylation and acts as a tether to facilitate autophagosome maturation.

Highlights d Seipin is enriched at ER-MAMs d Seipin interacts with MAM calcium regulators in a nutritionally regulated manner d Adipocyte seipin deficiency impairs mitochondrial calcium import and ATP production d Inducible seipin removal from adipose tissue leads to rapid mitochondrial dysfunction

The endoplasmic reticulum (ER) is comprised of a controlled ratio of sheets and tubules, which are maintained by several proteins with multiple functions. Reticulons (RTNs), especially RTN4, and DP1/Yop1p family members are known to induce ER membrane curvature. RTN4B is the main RTN4 isoform expressed in non-neuronal cells. In this study, we identified FAM134C as a RTN4B interacting protein in mammalian, non-neuronal cells. FAM134C localized specifically to the ER tubules and sheet edges. Ultrastructural analysis revealed that overexpression of FAM134C induced formation of unbranched, long tubules or dense globular structures comprised of heavily branched narrow tubules. In both cases, tubules were non-motile. ER tubulation was dependent on the reticulon homology domain (RHD) close to the N-terminus. FAM134C plays a role in the autophagy pathway as its level elevated significantly upon amino acid starvation but not during ER stress. Moreover, FAM134C depletion reduced the number and size of autophagic structures and the amount of ER as a cargo within autophagic structures under starvation conditions. Dominant-negative expression of FAM134C forms with mutated RHD or LC3 interacting region (LIR) also led to the reduced number of autophagic structures. Our results suggest that FAM134C provides a link between regulation of ER architecture and ER turnover by promoting ER tubulation required for subsequent ER fragmentation and engulfment into autophagosomes. [Media: see text] [Media: see text] [Media: see text] [Media: see text]

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The endoplasmic reticulum (ER) is a large, single‐copy, membrane‐bound organelle that comprises an elaborate 3D network of structurally diverse subdomains: highly curved tubules, flat sheets, and parts that form contacts with nearly every other organelle of the cell. The ER is essential for the synthesis, modification, and transport of membrane and secretory proteins; it is also the site of cytosolic calcium level regulation and synthesis and transport of several lipids. To accommodate the vast range of functions, the ER network spreads throughout the cell, and its functions are distributed into structural subdomains according to their specific requirements. Many structural determinants of the network formation and maintenance have been described; however, it is not yet understood e.g. how different functions are distributed to the various subdomains, why the ER is constantly rearranging its architecture, and how the sheet/tubule –balance is maintained. Proper ER operation requires an intricate balance within and in‐between dynamics, morphology, and functions. While ER structure is in constant flux, it does not move en masse, and the network movement is achieved through dynamics of individual subdomains and through network remodelling, which are accomplished through interactions with the cytoskeleton. Combining live cell imaging, thin section TEM and two 3D‐EM methods, we show that dynamic microtubules and actin filament arrays together contribute to the maintenance of ER sheet‐tubule balance. Perturbations of microtubule or actin cytoskeleton readily shift the balance towards tubules or sheets, which in turn can result in formation of sheet remnants or long and less fenestrated abnormal sheets. We recently identified the unconventional motor protein myosin 1c localizing to and regulating the ER associated actin filament arrays. Manipulation of myo1c levels disturbed the dynamics of actin arrays and affected ER sheet morphology similarly to actin manipulations with drugs. Tubular ER phenotype of myo1c‐depleted cells could be rescued with wild type myo1c, but not with a mutated form lacking the actin binding domain. We propose that ER ‐associated actin filaments have a role in maintaining the ER sheet‐tubule balance and sheet structure by regulating sheet remodeling events, and thus support the maintenance of sheets as a stationary subdomain of the otherwise dynamic ER network. Knowledge of the mechanisms behind the structural maintenance and dynamics will be the key towards deeper understanding of ER functions and their regulation, and eventually, in unravelling molecular mechanisms behind various ER‐associated diseases.