A third of the human genome encodes N-glycosylated proteins. These are co-translationally translocated into the lumen/membrane of the endoplasmic reticulum (ER) where they fold and assemble before they are transported to their final destination. Here, we show that calnexin, a major ER chaperone involved in glycoprotein folding is palmitoylated and that this modification is mediated by the ER palmitoyltransferase DHHC6. This modification leads to the preferential localization of calnexin to the perinuclear rough ER, at the expense of ER tubules. Moreover, palmitoylation mediates the association of calnexin with the ribosome-translocon complex (RTC) leading to the formation of a supercomplex that recruits the actin cytoskeleton, leading to further stabilization of the assembly. When formation of the calnexin-RTC supercomplex was affected by DHHC6 silencing, mutation of calnexin palmitoylation sites or actin depolymerization, folding of glycoproteins was impaired. Our findings thus show that calnexin is a stable component of the RTC in a manner that is exquisitely dependent on its palmitoylation status. This association is essential for the chaperone to capture its client proteins as they emerge from the translocon, acquire their N-linked glycans and initiate folding.

SARS-CoV-2 virions are surrounded by a lipid bilayer that contains membrane proteins such as spike, responsible for target-cell binding and virus fusion. We found that during SARS-CoV-2 infection, spike becomes lipid modified, through the sequential action of the S-acyltransferases ZDHHC20 and 9. Particularly striking is the rapid acylation of spike on 10 cytosolic cysteines within the ER and Golgi. Using a combination of computational, lipidomics, and biochemical approaches, we show that this massive lipidation controls spike biogenesis and degradation, and drives the formation of localized ordered cholesterol and sphingolipid-rich lipid nanodomains in the early Golgi, where viral budding occurs. Finally, S-acylation of spike allows the formation of viruses with enhanced fusion capacity. Our study points toward S-acylating enzymes and lipid biosynthesis enzymes as novel therapeutic anti-viral targets.

The anthrax toxin is a tripartite toxin, where the two enzymatic subunits require the third subunit, the protective antigen (PA), to interact with cells and be escorted to their cytoplasmic targets. PA binds to cells via one of two receptors, TEM8 and CMG2. Interestingly, the toxin times and triggers its own endocytosis, in particular through the heptamerization of PA. Here we show that PA triggers the ubiquitination of its receptors in a β-arrestin-dependent manner and that this step is required for clathrin-mediated endocytosis. In addition, we find that endocytosis is dependent on the heterotetrameric adaptor AP-1 but not the more conventional AP-2. Finally, we show that endocytosis of PA is strongly dependent on actin. Unexpectedly, actin was also found to be essential for efficient heptamerization of PA, but only when bound to one of its 2 receptors, TEM8, due to the active organization of TEM8 into actin-dependent domains. Endocytic pathways are highly modular systems. Here we identify some of the key players that allow efficient heptamerization of PA and subsequent ubiquitin-dependent, clathrin-mediated endocytosis of the anthrax toxin.

S-Palmitoylation is the only reversible post-translational lipid modification. Knowledge about the DHHC palmitoyltransferase family is still limited. Here we show that human ZDHHC6, which modifies key proteins of the endoplasmic reticulum, is controlled by an upstream palmitoyltransferase, ZDHHC16, revealing the first palmitoylation cascade. The combination of site specific mutagenesis of the three ZDHHC6 palmitoylation sites, experimental determination of kinetic parameters and data-driven mathematical modelling allowed us to obtain detailed information on the eight differentially palmitoylated ZDHHC6 species. We found that species rapidly interconvert through the action of ZDHHC16 and the Acyl Protein Thioesterase APT2, that each species varies in terms of turnover rate and activity, altogether allowing the cell to robustly tune its ZDHHC6 activity.

Loss-of-function mutations in capillary morphogenesis gene 2 (CMG2/ANTXR2), a transmembrane surface protein, cause hyaline fibromatosis syndrome (HFS), a severe genetic disorder that is characterized by large subcutaneous nodules, gingival hypertrophy and severe painful joint contracture. Here we show that CMG2 is an important regulator of collagen VI homoeostasis. CMG2 loss of function promotes accumulation of collagen VI in patients, leading in particular to nodule formation. Similarly, collagen VI accumulates massively in uteri of Antxr2−/− mice, which do not display changes in collagen gene expression, and leads to progressive fibrosis and sterility. Crossing Antxr2−/− with Col6a1−/− mice leads to restoration of uterine structure and reversion of female infertility. We also demonstrate that CMG2 may act as a signalling receptor for collagen VI and mediates its intracellular degradation.