Scyl1 scaffolds class II Arfs to selective subcomplexes of coatomer via the γ-COP appendage domain

Abstract

Coatomer (COPI)-coated vesicles mediate membrane trafficking in the early secretory pathway. There are at least three subclasses of COPI coats and two classes of Arf GTPases that couple COPI coat proteins to membranes. Whether mechanisms exist to link specific Arfs to specific COPI subcomplexes is unknown. We now demonstrate that Scy1-like protein 1 (Scyl1), a member of the Scy1-like family of catalytically inactive protein kinases, oligomerizes through centrally located HEAT repeats and uses a C-terminal RKXX-COO 2 motif to interact directly with the appendage domain of coatomer subunit c-2 (also known as COPG2 or c2-COP). Through a distinct site, Scyl1 interacts selectively with class II Arfs, notably Arf4, thus linking class II Arfs to c2-bearing COPI subcomplexes. Therefore, Scyl1 functions as a scaffold for key components of COPI coats, and disruption of the scaffolding function of Scyl1 causes tubulation of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and the cisGolgi, similar to that observed following the loss of Arf and Arf-guaninenucleotide-exchange factor (GEF) function. Our data reveal that Scyl1 is a key organizer of a subset of the COPI machinery.

Formation of COPI-coated vesicles at a glance

“…Fairly little is known regarding whether these coats perform specialized roles within mammalian cells and whether ArfGAP2 and ArfGAP3 are coupled to and regulated in a differential manner by these coatomer isoforms. Scyl1, a protein that regulates Golgi complex morphology (Burman et al, 2010), specifically binds the γ2 appendage domain of COPI (Hamlin et al, 2014). Scyl1 also selectively links Arf4 to γ2-containing coatomer isoforms.…”

Intermediate Compartment: A Sorting Station between the Endoplasmic Reticulum and the Golgi Apparatus

“…For example, incubation of cells at 15-16 1C inhibits the formation of IC tubules, but causes the expansion of the globular domain, while the shift of cells to 37 1C generates tubular networks containing both antero-and retrograde markers (Blum et al, 2000;Ben-Takaya et al, 2005;Simpson et al, 2006). Proliferation of the tubules is induced when COPI function is compromised (Szul et al, 2007;Marie et al, 2009;Ben-Takaya et al, 2010;Tomás et al, 2010;Hamlin et al, 2014), but also occurs under physiological conditions. In differentiating neuroendocrine PC12 cells the Rab1A-positive tubular IC domain expands and the tubules move from the cell body to the forming neurites accumulating in their growth cones (Sannerud et al, 2006; Figure 5).…”

“…Role of COPI coats: There are at least three subtypes of COPI coats (Beck et al, 2009), and four Arf GTPases that regulate their membrane binding (Popoff et al, 2011). Three Arfs appear to act at the ER-Golgi boundary and two of these associate with membranes in a BFA-resistant manner (Volpicelli-Daley et al, 2005;Chun et al, 2008;Duijsings et al, 2009;Ben-Tekaya et al, 2010;Hamlin et al, 2014), suggesting that different types of COPI vesicles mediate two-way trafficking at the level of the IC. The role of COPI in anterograde transport has been considered for some time (Hosobuchi et al, 1992;Pepperkok et al, 1993;Peter et al, 1993;Orci et al, 1997;Malsam et al, 2005).…”

“…The level of p58/ERGIC-53 mRNA is up-regulated by the unfolded protein response (UPR), which also requires yeast Ypt1 function, supporting a link between this signaling pathway and ER-Golgi trafficking (Nyfeler et al, 2003;Tsvetanova et al, 2012). Protein kinases Src, aPKC, and Scyl1 have been implicated in COPI function at the level of the IC (Tisdale and Artalejo, 2006;Hamlin et al, 2014) and PKC and its downstream effectors appear to control IC morphology (Ben-Tekaya et al, 2010;Sugawara et al, 2012). The activation of neuronal Trk receptor tyrosine kinases in the IC initiates signaling via the MEK pathway leading to Golgi fragmentation (Schecterson et al, 2010).…”

Golgi compartmentation and identity

“…This terminology comes from morphological studies of the plant secretory pathway [22], but studies of mammalian cells also provide evidence for two classes of COPI vesicles [54-57]. In mammalian cells, COPI subunit isoforms apparently interact with specific partners during different COPI-dependent pathways [58 •]. More generally, the properties of a COPI vesicle may be dictated by the composition of the parental compartment, with the same basic machinery generating either COPIa or COPIb vesicles at different stages of maturation.…”