The targeting of the oxysterol‐binding protein ORP3a to the endoplasmic reticulum relies on the plant VAP33 homolog PVA12

Abstract

SUMMARYIn plants, sterols play fundamental roles as membrane constituents in the biosynthesis of steroid hormones, and act as precursors for cell wall deposition. Sterols are synthesized in the endoplasmic reticulum (ER), but mainly accumulate in the plasma membrane. How sterols are trafficked in plant cells is largely unknown. In non-plant systems, oxysterol-binding proteins have been involved in sterol trafficking and homeostasis. There are at least twelve homologs of oxysterol-binding proteins in the Arabidopsis genome, but the biology of these proteins remains for the most part obscure. Here, we report our analysis of the targeting requirements and the sterol-binding properties of a small Arabidopsis oxysterol-binding protein, ORP3a. We have determined that ORP3a is a bona fide sterol-binding protein with sitosterol-binding properties. Live-cell imaging analyses revealed that ORP3a is localized at the ER, and that binding to this organelle depends on a direct interaction with PVA12, a member of the largely uncharacterized VAP33 family of plant proteins. Molecular modeling analyses and site-directed mutagenesis led to the identification of a novel protein domain that is responsible for the PVA12-ORP3a interaction. Disruption of the integrity of this domain caused redistribution of ORP3a to the Golgi apparatus, suggesting that ORP3a may cycle between the ER and the Golgi. These results represent new insights into the biology of sterol-binding proteins in plant cells, and elucidate a hitherto unknown relationship between members of oxysterol-binding protein and VAP33 families of plant proteins in the early plant secretory pathway.

Arabidopsis SYT1 maintains stability of cortical endoplasmic reticulum networks and VAP27-1-enriched endoplasmic reticulum–plasma membrane contact sites

“…All the 13 members of the Arabidopsis NET family contain a NET actin-binding (NAB) domain and various numbers of coiled-coil domains that can simultaneously interact with the actin filaments and different membrane compartments (Deeks et al , 2012; Hawkins et al , 2014; Wang et al , 2014). Arabidopsis VAP27 proteins belong to the VAP33-like family which are homologs of mammalian VAPs and yeast suppressor of choline sensitivity (Scs2) (Sutter et al , 2006; Saravanan et al , 2009). The conserved major sperm (MSP) domain is essential for VAP27-1 to anchor on the ER–PM contact sites and for interaction of VAP27-1 with NET3C.…”

Analysis of the Key Elements of FFAT-Like Motifs Identifies New Proteins That Potentially Bind VAP on the ER, Including Two AKAPs and FAPP2

“…FFAT-like motifs in ORPs show marginal difference from FFAT, and bind VAP tightly [23], [26], [30]. Although some VAP interactors, have no discernible motif like FFAT [31], [32], [33], [34], there are other examples of VAP interactors that have been suggested to contain FFAT-like motifs [35], [36], [37]. One is protrudin, an integral membrane protein that binds rab11 and migrates from the ER to endosomes, and which contains 1 EFKDA-E 7 [35], [38].…”

“…GLTP interacts directly with VAP, and a FFAT-like motif was identified in GLTP: 32 PFFDC-G 38 , which has the 2 FFD 4 core [37]. Similarly, Orp3a, one of 12 ORPs in Arabidopsis , binds VAP via a small region within the ORP domain that has features of the FFAT core: 2 WFD 4 [36]. In protrudin, GLTP and Orp3a, mutations in the identified FFAT cores inhibited function, suggesting that FFAT-like motifs can vary considerably from 1 EFFDA-E 7 and still be physiologically relevant.…”

Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

“…MSBP1 and MSBP2 can bind to progesterone, brassinolide, and stigmasterol with different affinities and presumably have a role in steroid signaling (Yang et al, 2005). PVA12 was demonstrated to be important for the ER localization of sterol binding proteins (Saravanan et al, 2009). SMT2 expression impacts sterol composition of the membrane (Schaeffer et al, 2001) and is associated with vascular patterning (Carland et al, 2002).…”