The E‐Syt3 cleavage and traffic uncovers the primordial cisterna, a new organelle that mothers the lipid droplets in the adipocyte

Extended synaptotagmins are endoplasmic reticulum proteins consisting of an SMP domain and multiple C2 domains that bind phospholipids and Ca2+. E‐Syts create contact junctions between the ER and plasma membrane (PM) to facilitate the exchange of glycerophospholipids between the apposed membranes. We find in the differentiating adipocyte that the E‐Syt3 carboxyl domain is cleaved by a multi‐step mechanism that includes removing the C2C domain. Confocal and live‐cell time‐lapse studies show that truncated E‐Syt3ΔC2C, as well as endogenous E‐Syt3 and the coat protein PLIN1, target the LDs from an annular, single giant ER cisterna. Inhibition of the proteasome blocks the proteolytic cleavage of Esyt3 and E‐Syt3ΔC2C and causes the E‐Syt3ΔC2C retention in the giant cisterna. The Esyt3 and PLIN1 distributions and LDs biogenesis show that the primordial cisterna, as we call it, is the birth and nurturing site of LDs in the adipocyte. Isoproterenol‐induced lipolysis results in loss of cytoplasmic LDs and reappearance of the primordial cisterna. Electron microscopy and 3D‐electron tomography studies show that the primordial cisterna consists of a tightly packed network of varicose tubules with extensively blistered membranes. Rounds of homotypic fusions from nascent to mature LDs play a central role in LD growth. The knockdown of E‐Syt3 inhibits LD biogenesis. The identification of the primordial cisterna, an organelle that substitutes the randomly scattered ER foci that mother the LDs in non‐adipose cells, sets the stage for a better understanding of LD biogenesis in the adipocyte. (A) Development of the primordial cisterna. The emergence of the primordial cisterna from the ER is likely to be intimately linked with the concentration of materials used in LD formation. The primordial cisterna assembly may involve protein‐protein and protein‐lipid interactions causing nucleation‐propagation effects. The concentration of E‐Syt3 (green) in the primordial cisterna requires the removal of the carboxyl C2C domain that anchors the ER protein to the plasma membrane. (B) Lipid droplet biogenesis and early growth in the primordial cisterna. The synthesis and coalescence of NLs (red) between the membrane leaflets of the cisterna create the lens‐shaped seeds for new LDs. Nascent LDs bud into the alveolar spaces produced by the cisterna fraying. In the alveoli, the new LDs grow by homotypic fusion. (C) LDs grow and maturation. Cytoplasmic LDs continue growing by successive round