Drosophila Sec16 Mediates the Biogenesis of tER Sites Upstream of Sar1 through an Arginine-Rich Motif

Abstract

tER sites are specialized cup-shaped ER subdomains characterized by the focused budding of COPII vesicles. Sec16 has been proposed to be involved in the biogenesis of tER sites by binding to COPII coat components and clustering nascent-coated vesicles. Here, we show that Drosophila Sec16 (dSec16) acts instead as a tER scaffold upstream of the COPII machinery, including Sar1. We show that dSec16 is required for Sar1-GTP concentration to the tER sites where it recruits in turn the components of the COPII machinery to initiate coat assembly. Last, we show that the dSec16 domain required for its localization maps to an arginine-rich motif located in a nonconserved region. We propose a model in which dSec16 binds ER cups via its arginine-rich domain, interacts with Sar1-GTP that is generated on ER membrane by Sec12 and concentrates it in the ER cups where it initiates the formation of COPII vesicles, thus acting as a tER scaffold.

Sec16A defines the site for vesicle budding from the endoplasmic reticulum on exit from mitosis

“…However, it does not preclude mitotic inhibition of interactions of Sec16 with downstream COPII components (AltanBonnet et al, 2006). Although the mechanisms underlying this inhibition remain unknown, these data also reinforce previous data showing that Sar1, Sec23-Sec24, Sec13-Sec31 all lie downstream of Sec16 (Ivan et al, 2008;Hughes et al, 2009). In fixed cells expressing Venus-Sec16AN, other COPII proteins, Sec24C ( Fig.…”

“…Lentiviral transduction of FP-Sec16A was precluded by persistent recombination of the parent vector during cloning. Importantly, Venus-Sec16AN includes both the central conserved domain and upstream sequences shown to be necessary for membrane association and targeting to ERES (Ivan et al, 2008;Hughes et al, 2009). Specifically, it colocalises exactly with endogenous Sec16A and localises adjacent to Sec24C and Sec31A labelling, as is seen for endogenous Sec16A ); its expression does not alter the expression of endogenous Sec16A, and it rescues the phenotypes of decreased ERES number and Golgi fragmentation caused by suppression of endogenous Sec16A (supplementary material Fig.…”

“…Together, these components form a vesicle coat that selects cargo for export, deforms the membrane, and ultimately generates coated transport vesicles following budding. An additional component, Sec16, potentiates budding (Supek et al, 2002) and appears to mark the transitional ER membrane and define the site of COPII vesicle budding (Connerly et al, 2005;Ivan et al, 2008;Hughes et al, 2009). Inhibition of vesicle budding from the ER during prometaphase (Farmaki et al, 1999) correlates with the loss of COPII proteins (Sec23-Sec24 and Sec13-31) from the ERES membrane (Farmaki et al, 1999;Hammond and Glick, 2000;Prescott et al, 2001;Stephens, 2003;Altan-Bonnet et al, 2006).…”

Regulation of Sec16 levels and dynamics links proliferation and secretion

“…Sec16 was shown to bind to ERES through an arginine-rich region without major help from co-factors (Ivan et al, 2008). Others have shown that Sec16 remains associated with ERES throughout mitosis without the presence of COPII (Hughes et al, 2009).…”

“…Sec16A (hereafter called Sec16) plays an important role in ERES homeostasis as it interacts with several COPII components and regulates their function (Bhattacharyya and Glick, 2007;Connerly et al, 2005;Farhan et al, 2008;Ivan et al, 2008;Supek et al, 2002;Watson et al, 2006;Espenshade et al, 1995;Gimeno et al, 1996;Whittle and Schwartz, 2010;Kung et al, 2012). Various post-translational modifications, such as phosphorylation (Farhan et al, 2010;Koreishi et al, 2013;Lord et al, 2011;Sharpe et al, 2011), ubiquitylation (Jin et al, 2012) or glycosylation (Dudognon et al, 2004), were reported to modulate ER export, but the molecular details and functional consequences of this regulation remain to be established.…”