ESCRT-III drives the final stages of CUPS maturation for unconventional protein secretion

Abstract

The unconventional secretory pathway exports proteins that bypass the endoplasmic reticulum. In Saccharomyces cerevisiae, conditions that trigger Acb1 secretion via this pathway generate a Grh1 containing compartment composed of vesicles and tubules surrounded by a cup-shaped membrane and collectively called CUPS. Here we report a quantitative assay for Acb1 secretion that reveals requirements for ESCRT-I, -II, and -III but, surprisingly, without the involvement of the Vps4 AAA-ATPase. The major ESCRT-III subunit Snf7 localizes transiently to CUPS and this was accelerated in vps4Δ cells, correlating with increased Acb1 secretion. Microscopic analysis suggests that, instead of forming intraluminal vesicles with the help of Vps4, ESCRT-III/Snf7 promotes direct engulfment of preexisting Grh1 containing vesicles and tubules into a saccule to generate a mature Acb1 containing compartment. This novel multivesicular / multilamellar compartment, we suggest represents the stable secretory form of CUPS that is competent for the release of Acb1 to cells exterior.DOI: http://dx.doi.org/10.7554/eLife.16299.001

Structures, Functions, and Dynamics of ESCRT-III/Vps4 Membrane Remodeling and Fission Complexes

“…These include the formation of CUPs (compartments for unconventional protein secretion), which are cup-shaped endoplasmic reticulum (ER)-derived vesicles involved in noncanonical protein secretion (Curwin et al 2016), and the invagination of ER membranes to form the replication compartments of some positive-stranded RNA viruses (Barajas et al 2009, Diaz et al 2015). …”

Reactive oxygen species (ROS) triggers unconventional secretion of antioxidants and Acb1

“…We employed a zymography-based assay to directly test whether secreted SOD1 is enzymatically active in yeast cells cultured in potassium acetate. A mild cell wall extraction assay developed to detect starvation specific secretion of Acb1 and SOD1 was performed in non-denaturing conditions (Curwin et al, 2016;Cruz-Garcia et al, 2017). Intracellular and secreted proteins were then separated by native-gel electrophoresis and a standardized in-gel SOD1 activity assay was performed as described previously (Beauchamp and Fridovich, 1971).…”

“…Do cells secrete other antioxidant enzymes, like SOD1, upon nutrient starvation? Wild type yeast were cultured in growth or starvation medium, the cell wall proteins were extracted under the conditions described previously (Curwin et al, 2016) and subsequently analysed by mass spectrometry. We also analysed cell wall proteins from cells lacking Vps23, which is required for starvation induced secretion of Acb1 and SOD1, to assist in identification of proteins exported by the same pathway.…”

“…1 C; Table S1). Secretion of SOD1 (and Acb1) requires Grh1 (Curwin et al, 2016;Cruz-Garcia et al, 2017), so we first tested the involvement of Grh1 in secretion of these enzymes. A secretion assay of wild-type and grh1D starved cells revealed that the release of Ahp1, Trx1 and Trx2 upon starvation is also Grh1 dependent ( Fig.…”

“…Fibroblast growth factor (FGF)2 and Acyl-CoA binding protein (AcbA/Acb1) are two of the best studied examples of unconventionally secreted proteins thus far. FGF2 follows a Type II route, whereas, Acb1 takes a Type III path via that involves a cellular compartment called CUPS, the cytoplasmic proteins Grh1 and a subset of ESCRTs (Duran et al, 2010;Cruz-Garcia et al, 2014;Steringer et al, 2015;Curwin et al, 2016;Steringer et al, 2017;Cruz-Garcia et al, 2018). More recently, the export of cytoplasmic enzyme superoxide dismutase 1 (SOD1) was reported to follow the same pathway as Acb1, and both depend on a di-acidic motif (Cruz-Garcia et al, 2017).…”