Spatial organization of phospholipid synthesis in eukaryotes is critical for cellular homeostasis. The synthesis of phosphatidylcholine (PC), the most abundant cellular phospholipid, occurs redundantly via the ER-localized Kennedy pathway and a pathway that traverses the ER and mitochondria via membrane contact sites. The basis of the ER-mitochondrial PC synthesis pathway is the exclusive mitochondrial localization of a key pathway enzyme, phosphatidylserine decarboxylase Psd1, which generates phosphatidylethanolamine (PE). We find that Psd1 is localized to both mitochondria and the ER. Our data indicate that Psd1-dependent PE made at mitochondria and the ER has separable cellular functions. In addition, the relative organellar localization of Psd1 is dynamically modulated based on metabolic needs. These data reveal a critical role for ER-localized Psd1 in cellular phospholipid homeostasis, question the significance of an ER-mitochondrial PC synthesis pathway to cellular phospholipid homeostasis, and establish the importance of fine spatial regulation of lipid biosynthesis for cellular functions.

Promoting axon regeneration in the central and peripheral nervous system is of clinical importance in neural injury and neurodegenerative diseases. Both pro- and antiregeneration factors are being identified. We previously reported that the Rtca mediated RNA repair/splicing pathway restricts axon regeneration by inhibiting the nonconventional splicing of Xbp1 mRNA under cellular stress. However, the downstream effectors remain unknown. Here, through transcriptome profiling, we show that the tubulin polymerization-promoting protein (TPPP) ringmaker/ringer is dramatically increased in Rtca-deficient Drosophila sensory neurons, which is dependent on Xbp1. Ringer is expressed in sensory neurons before and after injury, and is cell-autonomously required for axon regeneration. While loss of ringer abolishes the regeneration enhancement in Rtca mutants, its overexpression is sufficient to promote regeneration both in the peripheral and central nervous system. Ringer maintains microtubule stability/dynamics with the microtubule-associated protein futsch/MAP1B, which is also required for axon regeneration. Furthermore, ringer lies downstream from and is negatively regulated by the microtubule-associated deacetylase HDAC6, which functions as a regeneration inhibitor. Taken together, our findings suggest that ringer acts as a hub for microtubule regulators that relays cellular status information, such as cellular stress, to the integrity of microtubules in order to instruct neuroregeneration.

The endoplasmic reticulum (ER) supports biosynthesis of proteins with diverse transmembrane domain (TMD) lengths and hydrophobicity. Features in transmembrane domains such as charged residues in ion channels are often functionally important, but could pose a challenge during cotranslational membrane insertion and folding. Our systematic proteomic approaches in both yeast and human cells revealed that the ER membrane protein complex (EMC) binds to and promotes the biogenesis of a range of multipass transmembrane proteins, with a particular enrichment for transporters. Proximity-specific ribosome profiling demonstrates that the EMC engages clients cotranslationally and immediately following clusters of TMDs enriched for charged residues. The EMC can remain associated after completion of translation, which both protects clients from premature degradation and allows recruitment of substrate-specific and general chaperones. Thus, the EMC broadly enables the biogenesis of multipass transmembrane proteins containing destabilizing features, thereby mitigating the trade-off between function and stability.