A key player in the formation of endoplasmic reticulum sheets is CLIMP-63, but mechanistic details remained elusive. Here authors combined cellular experiments and mathematical modelling to show that S-acylation of CLIMP-63 regulates its function by mediating its oligomerisation, turnover, and localisation.

The complex architecture of the endoplasmic reticulum (ER) comprises distinct dynamic features, many at the nanoscale, that enable the coexistence of the nuclear envelope, regions of dense sheets and a branched tubular network that spans the cytoplasm. A key player in the formation of ER sheets is cytoskeleton-linking membrane protein 63 (CLIMP-63). The mechanisms by which CLIMP-63 coordinates ER structure remain elusive. Here, we address the impact of S-acylation, a reversible post-translational lipid modification, on CLIMP-63 cellular distribution and function. Combining native mass-spectrometry, with kinetic analysis of acylation and deacylation, and data-driven mathematical modelling, we obtain in-depth understanding of the CLIMP-63 life cycle. In the ER, it assembles into trimeric units. These occasionally exit the ER to reach the plasma membrane. However, the majority undergoes S-acylation by ZDHHC6 in the ER where they further assemble into highly stable super-complexes. Using super-resolution microscopy and focused ion beam electron microscopy, we show that CLIMP-63 acylation-deacylation controls the abundance and fenestration of ER sheets. Overall, this study uncovers a dynamic lipid post-translational regulation of ER architecture.