Disulfide bond formation in secretory proteins occurs primarily in the endoplasmic reticulum (ER), where multiple enzyme families catalyze cysteine cross-linking. Quiescin sulfhydryl oxidase 1 (QSOX1) is an atypical disulfide catalyst, localized to the Golgi apparatus or secreted from cells. We examined the physiological function for extracellular catalysis of de novo disulfide bond formation by QSOX1. QSOX1 activity was required for incorporation of laminin into the extracellular matrix (ECM) synthesized by fibroblasts, and ECM produced without QSOX1 was defective in supporting cell-matrix adhesion. We developed an inhibitory monoclonal antibody against QSOX1 that could modulate ECM properties and undermine cell migration.

The entry of calcium into mitochondria is central to metabolism, inter-organelle communication, and cell life/death decisions. Long-sought transporters involved in mitochondrial calcium influx and efflux have recently been identified. To obtain a unified picture of mitochondrial calcium utilization, a parallel advance in understanding the forms and quantities of mitochondrial calcium stores is needed. We present here the direct 3D visualization of mitochondrial calcium in intact mammalian cells using cryo-scanning transmission electron tomography (CSTET). Amorphous solid granules containing calcium and phosphorus were pervasive in the mitochondrial matrices of a variety of mammalian cell types. Analysis based on quantitative electron scattering revealed that these repositories are equivalent to molar concentrations of dissolved ions. These results demonstrate conclusively that calcium buffering in the mitochondrial matrix in live cells occurs by phase separation, and that solid-phase stores provide a major ion reservoir that can be mobilized for bioenergetics and signaling.

Background:The adenovirus E4orf4 protein must bind protein phosphatase 2A (PP2A) for its functions. Results: The E4orf4 binding site in PP2A was mapped to the ␣1,␣2 helices of the B55␣ subunit. Conclusion: The E4orf4 binding site in PP2A-B55␣ lies above the substrate binding site and does not overlap it. Significance: A novel functional significance was assigned to the ␣1,␣2 helices of the PP2A-B55␣ subunit.

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Quiescin sulfhydryl oxidase 1 (QSOX1) catalyzes the formation of disulfide bonds in protein substrates. Unlike other enzymes with related activities, which are commonly found in the endoplasmic reticulum, QSOX1 is localized to the Golgi apparatus or secreted. QSOX1 is upregulated in quiescent fibroblast cells and secreted into the extracellular environment, where it contributes to extracellular matrix assembly. QSOX1 is also upregulated in adenocarcinomas, though the extent to which it is secreted in this context is currently unknown. To achieve a better understanding of factors that dictate QSOX1 localization and function, we aimed to determine how post-translational modifications affect QSOX1 trafficking and activity. We found a highly conserved N-linked glycosylation site to be required for QSOX1 secretion from fibroblasts and other cell types. Notably, QSOX1 lacking a glycan at this site arrives at the Golgi, suggesting that it passes endoplasmic reticulum quality control but is not further transported to the cell surface for secretion. The QSOX1 transmembrane segment is dispensable for Golgi localization and secretion, as fully luminal and transmembrane variants displayed the same trafficking behavior. This study provides a key example of the effect of glycosylation on Golgi exit and contributes to an understanding of late secretory sorting and quality control.