Year of Award

2023

Document Type

Thesis

Degree Type

Master of Science (MS)

Degree Name

Cellular, Molecular and Microbial Biology

Department or School/College

Division Of Biological Sciences

Committee Chair

Jesse C. Hay

Commitee Members

Jesse C. Hay, Mark Grimes, Sarah Certel, Klara Briknarova

Keywords

ALG-2, Peflin, Calcium regulation, COPII, Cargo sorting, ER exit sites

Subject Categories

Cell and Developmental Biology | Cell Biology | Molecular Biology

Abstract

The ER-Golgi interface is a very dynamic environment that involves the movement of protein-loaded vesicles forward and backward. The movement of COPII-coated vesicles from the ER to the Golgi is the initial step in the movement of secreted, organellar, and cell surface proteins toward their final destinations. Many factors can regulate this step, including cytosolic calcium increases. In this study, we examined the effect of a transient calcium pulse on recruitment to ER exit sites of cargo proteins for ER export, the calcium-sensitive regulatory proteins apoptosis-linked gene 2 (ALG-2) and peflin, and the COPII outer coat subunits Sec31A and Sec13. We used immunofluorescence and live cell microscopy in normal rat kidney cells to monitor these events during and after a calcium surge induced by the ER calcium pump inhibitor 2,5-Di-(t-butyl)-1,4-hydroquinone (BHQ). We found that a calcium pulse can enhance the sorting of cargo proteins into ER exit sites for at least one hour following the pulse. This functional enhancement coincided with the recruitment of the outer coat proteins Sec13 and Sec31A to ER exit sites that persisted well beyond the calcium pulse. These functional and targeting changes were most likely directed by the calcium sensors ALG-2 and peflin, whose calcium-dependent kinetic recruitment patterns were also documented. One unexpected finding was that peflin recruitment to ER exit sites was increased by calcium; previous results had suggested that peflin would dissociate in response to calcium. Taken together our work demonstrates for the first time that a brief calcium event can initiate a cascade of functional and structural changes at ER exit sites that persists well beyond the period of elevated calcium. Such calcium-dependent regulation may ensure more efficient protein movement in the early secretory pathway after calcium-induced exocytosis of secretory vesicles or following the induction of cell proliferation or differentiation.

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