A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell-cell contacts.

Junctions between the ER and plasma membrane (PM) are implicated in calcium homeostasis, non-vesicular lipid transfer, and other cellular functions. Two ER proteins that function both as tethers to the PM via a polybasic C-terminus motif and as phospholipid transporters are brain-enriched TMEM24 (C2CD2L) and its paralog C2CD2. We report that both proteins also form a complex with band 4.

ATG9 vesicles comprise the seed membrane of mammalian autophagosomes.

As the autophagosome forms, its membrane surface area expands rapidly, while its volume is kept low. Protein-mediated transfer of lipids from another organelle to the autophagosome likely drives this expansion, but as these lipids are only introduced into the cytoplasmic-facing leaflet of the organelle, full membrane growth also requires lipid scramblase activity. ATG9 harbors scramblase activity and is essential to autophagosome formation; however, whether ATG9 is integrated into mammalian autophagosomes remains unclear.

Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], a phosphoinositide concentrated predominantly in the plasma membrane, binds endocytic clathrin adaptors, many of their accessory factors, and a variety of actin-regulatory proteins. Here we have used fluorescent fusion proteins and total internal reflection fluorescence microscopy to investigate the effect of acute PI(4,5)P(2) breakdown on the dynamics of endocytic clathrin-coated pit components and of the actin regulatory complex, Arp2/3. PI(4,5)P(2) breakdown was achieved by the inducible recruitment to the plasma membrane of an inositol 5-phosphatase module through the rapamycin/FRB/FKBP system or by treatment with ionomycin.

Rapid Ca2+-dependent decrease of protein ubiquitination at synapses.

Protein ubiquitination has been implicated in the regulation of axonal growth and synaptic plasticity as well as in the pathogenesis of neurodegenerative diseases. Here we show that depolarization-dependent Ca2+ influx into synaptosomes produces a global, rapid (range of seconds), and reversible decrease of the ubiquitinated state of proteins, which correlates with the Ca2+-dependent dephosphorylation of several synaptic proteins. A similar general decrease in protein ubiquitination was observed in nonneuronal cells on Ca2+ entry induced by ionomycin.