For a plasma membrane marker for use in total cell fluorescence calculations, pCAG-mGFP was purchased from Addgene (product: 14757). siRNA knockdowns and transfections For plasmid transfections, NRK or PC12?cells were transfected using Polyjet (SignaGen Laboratories), following the manufacturers instructions, about 24?h prior to transport assays. excitotoxicity and infection following decreases. In epithelial normal rat kidney (NRK) cells, the Ca2+-mobilizing agonist ATP causes ALG-2 to depress ER export, while in neuroendocrine PC12?cells, Ca2+ mobilization by ATP results in ALG-2-dependent enhancement of secretion. Furthermore, distinct Ca2+ signaling patterns in NRK cells produce opposing ALG-2-dependent effects on secretion. Mechanistically, ALG-2-dependent UAA crosslinker 1 hydrochloride 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. transport reconstitutions and results with purified ALG-2 have indicated that the protein is an inhibitor of vesicle budding or fusion (7, 17). On the other hand, some recent intact cell trafficking experiments indicate a suppressive role for ALG-2 based upon UAA crosslinker 1 hydrochloride ALG-2 depletion (18), while we implied a stimulatory role for ALG-2 because peflin suppressed transport by antagonizing stimulatory ALG-2-sec31A interactions (16). Furthermore, work on a presumed ALG-2 ortholog in yeast, Pef1p, demonstrated an inverse relationship, wherein Pef1p binding to the sec31 PRR was by Ca2+ and delayed coat recruitment to the membrane (19). A distinct line of investigation has suggested that sec31A function is regulated by mono-ubiquitination, and that both ALG-2 and peflin assist in the mono-ubiquitination through recruitment of the ubiquitin ligase CUL3KLHL12 to ERES, which was required for collagen secretion from osteosarcoma cells (20). Here we advance understanding of PEF protein secretory regulation by demonstrating that ALG-2 binding to ERES can stimulate inhibit ER-to-Golgi transport depending upon ALG-2:peflin expression ratios and the nature of Ca2+ signals. In response to short bursts of agonist-driven Ca2+ signaling, ALG-2 increases outer coat targeting to ERES and stimulates transport. This response could help stimulated cells proliferate and/or replenish Rabbit Polyclonal to PDXDC1 exhausted endocrine or exocrine secretory vesicles. On the other hand, a more relentless Ca2+ signal causes ALG-2 to markedly slow ER export. This novel physiological response that curtails COPII targeting could represent a protective mechanism against excitotoxicity or infection. Results Peflin expression levels determine ER-to-Golgi transport rates over a wide dynamic range in an ALG-2-dependent manner To investigate the dynamic range and functional interactions of PEF protein regulation of ER export, we forced individual, tandem, or reciprocal expression changes of the two proteins. Endogenous peflin and ALG-2 were either knocked down using transfection with siRNA or overexpressed by transfection with the wt, untagged rodent proteins in NRK cells. After 24?h of transfection, the initial rate of ER-to-Golgi transport of the synchronizable transmembrane protein cargo VSV-Gts045-GFP was determined by incubation for 10?min at the permissive temperature followed immediately by fixation and morphological quantitation of the ratio of VSV-G that has reached the Golgi remaining in the ER, as before (16). Figure?1columns 1 and 2 show that as previously reported (16), peflin knockdown significantly increased VGV-G transport above basal by 84%. On the other hand, overexpression of peflin (column 3) decreased transport by 23% below basal. Interestingly, the same two manipulations of ALG-2 expression (columns 4 and 5) caused little change in transport relative to basal, indicating that at steady state, peflin expression levels are more rate-limiting. Forced peflin overexpression and underexpression thus define a dynamic range of peflin regulation of transport at steady-state Ca2+ of 107% of basal secretory flux (84% above basal and 23% below) in UAA crosslinker 1 hydrochloride NRK cells. Open in a separate window Figure?1 Peflin expression levels define a wide dynamic range of trafficking effects in an ALG-2 dependent manner.indicate values for one-way ANOVA using the Dunnet post-hoc test, which compares each value with that of the control; ?contained dilutions of purified GST-peflin and GST-ALG-2 at a ratio that had been determined on Coomassie-stained gels to be equimolar to each other. The y-axis shows the band intensities of ALG-2 and peflin relative to their respective standards on the same exposure. From these values, the relative molar ratio of the two proteins was determined and is listed above each cell type. Image scale bar; 20?m. We next asked whether the effects of peflin overexpression and depletion depended upon the presence of.
