ALG-2 and Peflin Stimulate or Inffibit Copii Targeting and Secretion in Response to Calcium Signaling

Abstract

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.

ER-to-Golgi Transport in HeLa Cells Displays High Resilience to Ca2+ and Energy Stresses

“…In contrast to the luminal hGH construct, this construct contains a di-acidic ER export sequence displayed on the cytoplasmic surface of the ER, allowing active COPII sorting into vesicles. In line with our expectations and other reports [ 41 , 60 ], the transmembrane cargo construct was transported faster than the soluble cargo ( Figure 1 c). The ER-to-Golgi transport efficiency of the transmembrane cargo construct was also highly heterogeneous among individual HeLa cells.…”

“…To better assess the ER-to-Golgi transport activities of soluble and transmembrane constructs in HeLa cells, we additionally expressed the same constructs in NRK cells and visualized their transport. As compared to HeLa cells, similar transport efficiencies of both cargo constructs were observed in NRK cells, which represent a well-characterized cell model for high secretory transport efficiency [ 41 , 61 , 62 ] ( Supplementary Figure S2 ). Interestingly, in contrast to HeLa cells ( Figure 1 ), the mCherry-Golgi-7 construct expressed in NRK cells ( Supplementary Figure S3a ) stained several distributed small vesicular structures likely representing the trans-Golgi network.…”

“…To study ER-to-Golgi transport of a classical bulk-flow cargo, a fusion construct consisting of an ER-targeting signal sequence, GFP, and CADs, as well as human growth hormone at the C-terminus, was imaged. To visualize the transport of a transmembrane cargo, the transmembrane domain of vesicular stomatitis virus G protein (VSVG) coupled to the C-terminus of GFP and CADs was used, working as an ER export signal, as well as a membrane anchor [ 41 ]. The co-expression of the green fluorescent cargo fusion construct with mCherry-Golgi-7, a red fluorescent marker of the Golgi complex, was used to co-localize the cargo construct with its primary target organelle, i.e., the Golgi apparatus ( Figure 1 a and Supplementary Figure S1 ).…”

“…635045, Takarabio, 78100 Saint-Germain-en-Laye, France), the protein aggregates disaggregated and transport was initiated. Constructs are described in more detail in Sargeant et al 2020 [ 41 ]. The cells were co-transfected with one of the mentioned transport constructs and mCherry-Golgi-7 (excited at 561 nm), which helped define the Golgi apparatus location in the transport quantification.…”

“…At least in part, due to genetically encoded tools enabling transport visualization and subcellular Ca 2+ measurements, recent data have confirmed the importance of global and local Ca 2+ signals in regulating membrane trafficking for ER-to-Golgi transport, as well as exocytosis [ 41 , 42 , 43 , 44 ]. Since Ca 2+ storage and extrusion is dependent on the amount of available ATP [ 45 , 46 ], any changes in metabolic activity can consequently impact the secretory pathway.…”