Identifying a novel functional domain within the p115 tethering factor required for Golgi ribbon assembly and membrane trafficking

Abstract

SummaryThe tethering factor p115 (known as Uso1p in yeast) has been shown to facilitate Golgi biogenesis and membrane traffic in cells in culture. However, the role of p115 within an intact animal is largely unknown. Here, we document that depletion of p115 by using RNA interference (RNAi) in C. elegans causes accumulation of the 170 kD soluble yolk protein (YP170) in the body cavity and retention of the yolk receptor RME-2 in the ER and the Golgi within oocytes. Structure-function analyses of p115 have identified two homology regions (H1 and H2) within the N-terminal globular head and the coiled-coil 1 (CC1) domain as essential for p115 function. We identify a new C-terminal domain of p115 as necessary for Golgi ribbon formation and cargo trafficking. We show that p115 mutants that lack the fourth CC domain (CC4) act in a dominant-negative manner to disrupt Golgi and prevent cargo trafficking in cells containing endogenous p115. Furthermore, using RNAi of p115 and the subsequent transfection with p115 deletion mutants, we show that CC4 is necessary for Golgi ribbon formation and membrane trafficking in cells depleted of endogenous p115. p115 has been shown to bind a subset of ER-Golgi SNAREs through CC1 and CC4 domains (Shorter et al., 2002). Our findings show that CC4 is required for p115 function, and suggest that both the CC1 and the CC4 SNARE-binding motifs participate in p115-mediated membrane tethering.

Aβ-induced Golgi fragmentation in Alzheimer’s disease enhances Aβ production

“…This does not seem to be the cause in our model systems, given that we detected no reduction in protein levels of p115 or other Golgi structural proteins that we evaluated. Similar to GM130, p115 is involved in membrane tethering; disruption of p115 function also reduces membrane trafficking (42,43). However, our results using VSV-G as a marker demonstrated accelerated protein trafficking in cells with fragmented Golgi, whereas rescue of the Golgi structure by GRASP expression reduced protein trafficking (Fig.…”

Caspases rule the intracellular trafficking cartel

“…This key vesicle tethering factor is involved in COPI vesicle transport and in maintaining Golgi ribbon architecture. Indeed, interfering with p115 functions using antibodies or siRNAs results in Golgi fragmentation [51,52]. p115 mediates its tethering function by binding to SNAREs via four C-terminal CC domains and to both GM130 and giantin golgins via an acidic C-terminal domain.…”

“…The cleavage site is located following the second CC domain, thus, removing CC3, CC4, and the acidic domain required for golgins binding and vesicle docking. Moreover, expression of the N-terminal or C-terminal fragment mimicking the cleavage product is sufficient to induce Golgi disruption [37,52].…”

Intracellular trafficking and secretion of inflammatory cytokines

“…Golgins interact with the microtubule cytoskeleton and with many other proteins, including Rabs, to mediate aspects of anterograde and retrograde trafficking through the Golgi complex [69]. Golgins are also fundamental to maintaining the structure of the Golgi itself [70,71], and depletion of the TGN GRIP-golgin, GCC185, causes fragmentation of the Golgi ribbon and dispersal of the individual stacks [72]. TGN-based GRIP-golgins, p230/golgin-245 and golgin-97, have established roles in forming distinct tubulovesicular carriers for TNF, IL-6 and IL-10 trafficking in macrophages [18,54].…”