Post-translational Regulation of the Early Secretory Pathway

Abstract

The essential and broadly conserved coat protein 2 (COPII) machine mediates the first stage of protein secretion. This places COPII at the center of a eukaryotic cell system responsible for the accurate packaging, transport, and localization of one-third of the entire proteome – including proteins destined for Golgi membrane incorporation, endosomes, the plasma membrane, and extracellular secretion. The coat is composed of five core proteins – Sar1 (a GTPase), Sec23/Sec24 (the inner coat components), and Sec13/Sec31 (the outer coat). In addition to these, Sec12 and Sec16 are essential in vivo, and recent work has also established cTAGE5/Tango1 as essential for specific transport needs. Effective and regulated COPII transport requires careful spatial and temporal coordination of all these proteins, not least of which is Sec24, the cargo adapter of COPII. As the subunit directly implicated in interaction with transmembrane and lumenal cargo (through interactions with various transmembrane adapter proteins), Sec24 packages cargo selectively. In vertebrates, gene duplication of the ancestral Sec24 homolog has led to four paralogs – Sec24A, B, C, and D. Though each paralog interacts with a wide array of cargo, minor differences in their binding pockets confer some specificity for distinct cargo motifs. Nevertheless, a growing body of research suggests that the unique functions of these paralogs can be mostly attributed to differences in their tissue-specific expression. Tissue-specific expression of paralogs provides one mode for regulation of COPII transport. However, individual cells must be able to rapidly respond to stimuli and injury, requiring rapid changes to protein load and transport needs. Regulation at the level of transcription is not sufficient for this, leaving a significant gap in the field’s understanding of this essential stage of the secretory pathway. Recent work has revealed that the subunits of the COPII coat, including the Sec24 paralogs, are subject to extensive post-translational modification (PTM). These modifications, including phosphorylation and O-GlcNAcylation (modification by O-linked β-N-acetylglucosamine), regulate multiple aspects of COPII behavior, including uncoating, interaction with binding partners, and transport of specific cargo. However, the regulatory functions of identified PTMs on Sec24 paralogs have yet to be systematically described. Here, I characterize the function of phosphorylation and O-GlcNAcylation on the Sec24C paralog in human cells, primarily as these PTMs relate to regulation of COPII localization and transport in response to the cell cycle. This work significantly adds to previous literature which had reported that Sec24C is reciprocally modified by O-phosphate and O-GlcNAc in mitosis versus interphase, respectively. Advances in CRISPR-tagging technology allowed for characterization of Sec24C PTMs from the protein expressed at its endogenous locus and levels in HeLa cells. With this tool in hand, we discovered novel Sec24C PTM-sites by liquid-chromatography tandem mass spectrometry (LC-MS/MS), including a site (T214) that is preferentially phosphorylated in mitosis. Additionally, we identified three simultaneously modified phosphosites in the helical domain of Sec24C that modulate both its localization and stability. Extensive immunofluorescence (IF) studies combined with immunoblot (IB) analysis suggested that while O-GlcNAcylation may modulate the precise timing of mitosis-associated COPII dispersal, the modification is ultimately dispensable for this phenomenon to occur. However, results from Phos-Tag SDS-PAGE / IB experiments support the possibility that mitosis-associated phosphorylation of Sec24C, which appears broadly conserved across human tissue types, may be required for its mitotic dispersal, and consequentially, for the proper inheritance of endomembrane components from mother to daughter cells. In addition to these findings, this work assesses the function of the Sec24C paralog in the transport of specific cargo utilizing the retention using selective hooks (RUSH) assay, and consequentially identifying a counterintuitive inverse relationship between Sec24C level and the transport of both α-Mannosidase II (ManII), and secreted soluble horseradish peroxidase (ssHRP). Lasty, this work utilized additional IF and IP/IB experiments to address outstanding questions regarding the function of PTMs on COPII. Sec31A O-GlcNAcylation was found to impact its interaction with a binding partner, KLHL12. Preliminary studies implicated glucose starvation, which may decrease Sec24C O-GlcNAcylation, in the regulation of ERES and COPII dynamics and assembly. Additionally, I assessed whether the COPII-perturbing actions of the small molecules H89 and nocodazole might be correlated to changes in Sec24C PTM status, and identified that the kinase inhibitor, H89, significantly reduces Sec24C O-GlcNAcylation while simultaneously increasing its phosphorylation and affecting its localization in a previously uncharacterized manner. These data add to the growing body of evidence that PTMs serve as a rapid and stimulus-sensitive means to regulate the biochemical and cellular activities of COPII subunits, and consequentially, secretory transport. Future research should continue to build off this work and the tools I developed to more completely and systematically characterize the function of site-specific modifications on the regulation of Sec24C, particularly as they relate to the cell cycle dynamics and mitotic inheritance of COPII.