Golgi localization of oxysterol binding protein-related protein 4L (ORP4L) is regulated by ligand binding

Abstract

Oxysterol binding protein (OSBP)-related protein 4L (ORP4L, also known as OSBPL2), a closely related paralogue and interacting partner of OSBP, binds sterols and phosphatidylinositol 4-phosphate [PI(4)P] and regulates cell proliferative signalling at the plasma membrane (PM). Here, we report that ORP4L also interacts with the -Golgi network (TGN) in an OSBP-, sterol- and PI(4)P-dependent manner. Characterization of ORP4L lipid and VAP binding mutants indicated an indirect mechanism for translocation to ER-Golgi contact sites in response to 25-hydroxycholesterol that was dependent on OSBP and PI(4)P. shRNA silencing revealed that ORP4L was required to maintain the organization and PI(4)P content of the Golgi and TGN. In contrast, the interaction of ORP4L with the PM was not dependent on its sterol, PI(4)P or VAP binding activities. At the PM, ORP4L partially localized with a genetically encoded probe for PI(4)P but not with a probe for phosphatidylinositol 4,5-bisphosphate. We conclude that ORP4L is differentially localized to the PM and ER-Golgi contacts sites. OSBP-, lipid- and VAP-regulated interactions of ORP4L with ER-Golgi contact sites are involved in the maintenance of Golgi and TGN structure.

Heterologous expression and functional characterization of the ligand-binding domain of oxysterol-binding protein from Aspergillus oryzae

“…Human OSBP1 is a cholesterol-sensing regulator of two protein phosphatases: PTPPBS and the serine/ threonine phosphatase PP2A [16]. The mammalian ORP4L binds sterols and PI [4] P and regulates cell proliferative signaling at the plasma membrane [17], meanwhile it also plays a part in the maintenance of Golgi/TGN structure by means of a PI(4)P-dependent mechanism [8]. In yeast, Osh proteins (Osh3-Osh7) serve as controllers of PI [4] P signaling and metabolism [18], and Osh4 mediates sterol transport from ER to mitochondria [19].…”

“…OSBP and ORPs possess multiple targeting motifs and are localized to membrane contact sites between organelles. For instance, ORP4L was located to contact sites between endoplasmic reticulum (ER) and Golgi apparatus [8], and mammalian ORP1 and ORP3 localize to ER-Golgi and ER-plasma membrane (PM) contact sites, respectively [9,10]. A common feature of all ORPs is that ORPs share a conserved OSBPrelated ligand-binding domain (ORD) in their C-terminal regions.…”

Phosphorylation of a serine/proline-rich motif in oxysterol binding protein-related protein 4L (ORP4L) regulates cholesterol and vimentin binding

“…ORP4L has a complex distribution in cells, partitioning between the cytoplasm, ER, Golgi apparatus and PM [14, 15, 25]. In addition, N-terminal truncated ORP4S and ORP4M do not associate with the ER/Golgi/PM but instead interact with the vimentin intermediate filament network, causing it to bundle and aggregate in the perinuclear region [10].…”

“…Mechanistically, ORP4L binds PI(4,5)P 2 at the PM and presents the substrate to PLCβ3 for hydrolysis [13]. ORP4L is also localized to the Golgi apparatus by a sterol/OSBP-dependent mechanism and is involved in the maintenance of organelle structure and PI(4)P content [14]. As well, ORP4S and ORP4M constitutively aggregate vimentin intermediate filaments, and the ORP4 ORD binds vimentin in vitro [15].…”

Lipid Exchangers: Cellular Functions and Mechanistic Links With Phosphoinositide Metabolism

“…However, ORP4 has distinct functions, and it is unclear whether it acts as a sterol/PI(4)P exchanger. ORP4L can partially localize to ER-Golgi contact sites, with its PH domain and through heterodimerization with OSBP to regulate Golgi PI(4)P homeostasis ( Pietrangelo and Ridgway, 2018 ). However, ORP4 and shorter variants can interact with intermediate filaments called vimentin and remodel the vimentin network near the nucleus ( Wang et al, 2002 ).…”