Background: Whether ER to Golgi transport requires calcium, the source of calcium, and its mechanism is unknown. Results: A requirement for luminal calcium is demonstrated, and evidence is presented for a molecular effector pathway. Conclusion: Luminal calcium may regulate transport by activating these protein interactions. Significance: The described calcium effector pathway may lead to greater insight into calcium action at multiple transport steps.

Previous studies show that the COPII coat is phosphorylated. The phosphorylated coat, however, cannot rebind to the ER to initiate a new round of vesicle budding. The present study shows that Sit4p/PP6, a Ser/Thr phosphatase, dephosphorylates the COPII coat. Consistent with a role in coat recycling, Sit4p/PP6 regulates ER-to-Golgi traffic.

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.

The Golgi complex is the Grand Central Station of intracellular membrane trafficking in the secretory and endocytic pathways. Anterograde and retrograde export of cargo from the Golgi complex involves a complex interplay between the formation of coated vesicles and membrane tubules, although much less is known about tubule-mediated trafficking. Recent advances using in vitro assays have identified several cytoplasmic phospholipase A2 (PLA2) enzymes that are required for the biogenesis of membrane tubules and their roles in the functional organization of the Golgi complex. In this chapter we describe methods for the cell-free reconstitution of PLA2-dependent Golgi membrane tubule formation. These methods should facilitate the identification of other proteins that regulate this process.