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.

ER-to-Golgi transport is the first step in the constitutive secretory pathway, which, unlike regulated secretion, is believed to proceed nonstop independent of Ca 2+ flux. However, here we demonstrate that penta-EF hand (PEF) proteins ALG-2 and peflin constitute a hetero-bifunctional COPII regulator that responds to Ca 2+ signaling by adopting one of several distinct activity states. Functionally, these states can adjust the rate of ER export of COPII-sorted cargos up or down by ∼50%. We found that at steady-state Ca 2+ , ALG-2/peflin hetero-complexes bind to ER exit sites (ERES) through the ALG-2 subunit to confer a low, buffered secretion rate, while peflin-lacking ALG-2 complexes markedly stimulate secretion. Upon Ca 2+ signaling, ALG-2 complexes lacking peflin can either increase or decrease the secretion rate depending on signaling intensity and duration—phenomena that could contribute to cellular growth and intercellular communication following secretory increases or protection from excitotoxicity and infection following decreases. In epithelial normal rat kidney (NRK) cells, the Ca 2+ -mobilizing agonist ATP causes ALG-2 to depress ER export, while in neuroendocrine PC12 cells, Ca 2+ mobilization by ATP results in ALG-2-dependent enhancement of secretion. Furthermore, distinct Ca 2+ signaling patterns in NRK cells produce opposing ALG-2-dependent effects on secretion. Mechanistically, ALG-2-dependent depression of secretion involves decreased levels of the COPII outer shell and increased peflin targeting to ERES, while ALG-2-dependent enhancement of secretion involves increased COPII outer shell and decreased peflin at ERES. These data provide insights into how PEF protein dynamics affect secretion of important physiological cargoes such as collagen I and significantly impact ER stress.

Penta EF-hand (PEF) proteins apoptosis-linked gene 2 (ALG-2) and peflin are cytoplasmic Ca 2+ sensors for which physiological roles are still emerging. Here we demonstrate that adjustment of the ALG-2:peflin expression ratio can modulate basal ER export rates up or down by 107% of the basal rate. Through their ALG-2 subunit, ALG-2-peflin hetero-oligomers are shown to bind ERES and inhibit ER export of multiple cargo types, including collagen I, while ALG-2 by itself binds ERES to stimulate cargo sorting and ER export. During sustained Ca 2+ signaling, peflin and ALG-2 are demonstrated to sharply lower ER export rates by 40% in a novel secretory response to demanding physiological conditions. However, in highly Ca 2+stressed cells, peflin's suppressive role appears to promote pro-apoptotic unfolded protein response (UPR) signaling, indicating that the regulation is not necessarily pro-survival. Thus, regulation of secretion by PEF protein sub-complex binding to ERES in response to Ca 2+ signals impacts key cellular decision processes relevant to aging, neurodegeneration and diabetes.

Constitutive vesicle trafficking is the default pathway used by all cells for movement of intracellular cargoes between subcellular compartments and in and out of the cell. Classically, constitutive trafficking was thought to be continuous and unregulated, in contrast to regulated secretion, wherein vesicles are stored intracellularly until undergoing synchronous membrane fusion following a Ca 2+ signal. However, as shown in the literature reviewed here, many continuous trafficking steps can be up- or down-regulated by Ca 2+ , including several steps associated with human pathologies. Notably, we describe a series of Ca 2+ pumps, channels, Ca 2+ -binding effector proteins, and their trafficking machinery targets that together regulate the flux of cargo in response to genetic alterations as well as baseline and agonist-dependent Ca 2+ signals. Here, we review the most recent advances, organized by organellar location, that establish the importance of these components in trafficking steps. Ultimately, we conclude that Ca 2+ regulates an expanding series of distinct mechanistic steps. Furthermore, the involvement of Ca 2+ in trafficking is complex. For example, in some cases, the same Ca 2+ effectors regulate surprisingly distinct trafficking steps, or even the same trafficking step with opposing influences, through binding to different target proteins.

A questionnaire mailed to 65 workshop participants yielded a response rate of 46% (n = 30). Of the 30 respondents, 27% (n = 8) had bought new hardware or software because of attending the workshops, with the most common purchase being a new computer. Fifty-seven percent (n = 17) had increased their use of computers, with the most common applications being use of the Internet for information retrieval and electronic mail.