The yeast lgl family member Sro7p is an effector of the secretory Rab GTPase Sec4p

Abstract

Rab guanosine triphosphatases regulate intracellular membrane traffic by binding specific effector proteins. The yeast Rab Sec4p plays multiple roles in the polarized transport of post-Golgi vesicles to, and their subsequent fusion with, the plasma membrane, suggesting the involvement of several effectors. Yet, only one Sec4p effector has been documented to date: the exocyst protein Sec15p. The exocyst is an octameric protein complex required for tethering secretory vesicles, which is a prerequisite for membrane fusion. In this study, we describe the identification of a second Sec4p effector, Sro7p, which is a member of the lethal giant larvae tumor suppressor family. Sec4-GTP binds to Sro7p in cell extracts as well as to purified Sro7p, and the two proteins can be coimmunoprecipitated. Furthermore, we demonstrate the formation of a ternary complex of Sec4-GTP, Sro7p, and the t-SNARE Sec9p. Genetic data support our conclusion that Sro7p functions downstream of Sec4p and further imply that Sro7p and the exocyst share partially overlapping functions, possibly in SNARE regulation.

Yeast and fungal morphogenesis from an evolutionary perspective

“…Recently, Sro7 and Sro77, members of the conserved Lethal Giant Larvae (Lgl) family, were shown to play an important role in SNARE regulation. Sro7 and Sro77 interact with the exocyst and Sec4-GTP, as well as the t-SNARE Sec9 [58][59][60]. A new structure study suggests that Sro7 allosterically regulates the formation of the SNARE complex through its interaction with Sec9 [61].…”

In Vitro Reconstitution of Rab GTPase-dependent Vesicle Clustering by the Yeast Lethal Giant Larvae/Tomosyn Homolog, Sro7

“…Protein Purification-Sro7 with an N-terminal Protein A tag was obtained from a modification of the purification protocol described previously (2). Sro7 was expressed behind an ADH1 promoter from a high copy plasmid in a yeast pep4⌬ background strain.…”

“…The region bound by the tail was shown to overlap with the binding site of the plasma membrane t-SNARE Sec9 in a way that suggested the possibility of a "triggered" release of the SNARE by Sro7 (5). Although the Sec4 GTPase remained an attractive candidate triggering factor, its association with Sro7 had previously been shown to have no effect on Sro7's association with Sec9 (2). Therefore, the precise role of Sro7 as an effector of the Rab GTPase Sec4 remains elusive.…”

“…Loss of Sro7 and its redundant paralog Sro77 results in both cold-sensitive growth and a severe secretory defect in post-Golgi vesicle tethering/fusion with the plasma membrane (1). Importantly, Sro7 has been suggested to function as a direct effector of the GTPase Sec4 in a pathway that genetically appears to be parallel to that of the other effector of Sec4, the exocyst tethering complex (2). Although Sro7 has been found to interact with several components of the exocytic tethering and fusion apparatus (1,3,4), the precise molecular mechanism by which it functions in this process remains elusive.…”

Phosphorylation of the Rab exchange factor Sec2p directs a switch in regulatory binding partners

“…It plays an essential role at this stage of the secretory pathway by recruiting at least three different effectors. In its GTP-bound form, Sec4p binds directly to Myo2p, a type V myosin, on secretory vesicles, to promote their transport along polarized actin cables, to Sec15p, a component of the exocyst complex that tethers secretory vesicles to the plasma membrane in preparation for exocytic fusion, and to Sro7p, an Lgl family member shown to regulate exocytic SNARE function (3)(4)(5). The ability of Sec4p to interact with these effectors depends upon its activation by its GEF, Sec2p (6).…”