Luminal calcium regulates vesicle transport early in the secretory pathway. In ER-to-Golgi transport, depletion of luminal calcium leads to significantly reduced transport and a buildup of budding and newly budded COPII vesicles and vesicle proteins. Effects of luminal calcium on transport may be mediated by cytoplasmic calcium sensors near ER exits sites (ERES). The penta-EF-hand (PEF) protein apoptosis-linked gene 2 (ALG-2) stabilizes sec31A at ER exit sites (ERES) and promotes the assembly of inner and outer shell COPII components. However, in vitro and intact cell approaches have not determined whether ALG-2 is a negative or positive regulator, or a regulator at all, under basal physiological conditions. ALG-2 interacts with another PEF protein, peflin, to form cytosolic heterodimers that dissociate in response to calcium. However, a biological function for peflin has not been demonstrated and whether peflin and the ALG-2/peflin interaction modulates transport has not been investigated. Using an intact, single cell, morphological assay for ER-to-Golgi transport in normal rat kidney (NRK) cells, we found that depletion of peflin using siRNA resulted in significantly faster transport of the membrane cargo VSV-G. Double depletion of peflin and ALG-2 blocked the increased transport resulting from peflin depletion, demonstrating a role for ALG-2 in the increased transport. Furthermore, peflin depletion caused increased targeting of ALG-2 to ERES and increased ALG-2/sec31A interactions, suggesting that peflin may normally inhibit transport by preventing ALG-2/sec31A interactions. This work identifies for the first time a clear steady state role for a PEF protein in ER-to-Golgi transport—peflin is a negative regulator of transport.

Efforts to decrease the adverse effects of nuclear receptor (NR) drugs have yielded experimental agonists that produce better outcomes in mice. Some of these agonists have been shown to cause different, not just less intense, on-target transcriptomic effects; however, a structural explanation for such agonist-specific effects remains unknown. Here, we show that partial agonists of the NR peroxisome proliferator-associated receptor γ (PPARγ), which induce better outcomes in mice compared to clinically utilized type II diabetes PPARγ-binding drugs thiazolidinediones (TZDs), also favor a different group of coactivator peptides than the TZDs. We find that PPARγ full agonists can also be biased relative to each other in terms of coactivator peptide binding. We find differences in coactivator–PPARγ bonding between the coactivator subgroups which allow agonists to favor one group of coactivator peptides over another, including differential bonding to a C-terminal residue of helix 4. Analysis of all available NR–coactivator structures indicates that such differential helix 4 bonding persists across other NR–coactivator complexes, providing a general structural mechanism of biased agonism for many NRs. Further work will be necessary to determine if such bias translates into altered coactivator occupancy and physiology in cells.