A GPI Signal Peptide-Anchored Split-Ubiquitin (GPS) System for Detecting Soluble Bait Protein Interactions at the Membrane

Abstract

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Unusual Roles of Secretory SNARE SYP132 in Plasma Membrane H+-ATPase Traffic and Vegetative Plant Growth

“…The Qa-SNARE HabcΔ or the so-called Sp3 fragments retain the interactive surfaces associated with cognate partner binding; however, they lose interactions with high-M r regulatory proteins and ion transporters (Kargul et al, 2001). We used the yeast GPI signal peptide-anchored split-ubiquitin (GPS) system, which allows detection of soluble bait protein binding (Zhang et al, 2018), to test interactions of SYP132 HabcΔ . We observed that SYP132 HabcΔ retains one-to-one binding with the cognate SNAREs SNAP33 or VAMP721 and with the full-length SYP132 (Supplemental Fig.…”

“…Protein-protein interactions were done for SYP132 ΔC and SYP132 HabcΔ as baits in vector pEXG2Met-Dest and for SYP132, VAMP721, and SNAP33 as prey in vector pNX35-Dest (Grefen et al, 2009). The yeast GPS assay was performed as described (Zhang et al, 2018). The bait constructs were transferred in yeast strain THY.AP4, and prey constructs were transferred in THY.AP5.…”

K+ Channel-SEC11 Binding Exchange Regulates SNARE Assembly for Secretory Traffic

“…Therefore, we examined channel binding with these proteins. For mbSUS assays with the otherwise soluble baits SNAP33 and SEC11, we incorporated a glycosylphosphatidylinositol (GPI) anchor (Zhang et al, 2018) to express the corresponding protein fusions with NubG-X fusions of the full-length and truncated channel preys. Again, we recovered growth of the diploid yeast with the KC1 truncations, provided that residues 65 to 89 were present in each of the Nub-K 1 channel fusions (Fig.…”

“…A to C, Yeast mating-based split-ubiquitin assay for interaction of the VAMP721, SNAP33, and SEC11 fusions with KC1 and with Ala substitutions of key residues at the cytosolic face of the VSD as bait with Y-Cub fusions. VAMP721, SNAP33, and SEC11 as NubG-X prey fusions and with SNAP33 and SEC11 anchored via the GPI signal peptide (Zhang et al, 2018). Positive and negative controls are included in Supplemental Figure S1.…”

“…For split-ubiquitin assays and expression in Xenopus laevis oocytes, entry vector constructs of SYP121, SYP121 DC , VAMP721, VAMP721 DC , SNAP33, and SEC11, as well as KC1 and KAT1, their truncations, and single-site mutations were generated in pDONR207 as described previously (Grefen et al, 2010(Grefen et al, , 2015Zhang et al, 2015Zhang et al, , 2018Zhang et al, , 2019Lefoulon et al, 2018) and used in Gateway reactions with LR Clonase II (Life Technologies) to generate the corresponding destination constructs. Single-site mutations were generated by site-directed mutagenesis (Karnik et al, 2013a;Grefen et al, 2015;Zhang et al, 2017).…”