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Ligand-dependent localization and function of ORP–VAP complexes at membrane contact sites

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Abstract

Oxysterol-binding protein/OSBP-related proteins (ORPs) constitute a conserved family of sterol/phospholipid-binding proteins with lipid transporter or sensor functions. We investigated the spatial occurrence and regulation of the interactions of human OSBP/ORPs or the S. cerevisiae orthologs, the Osh (OSBP homolog) proteins, with their endoplasmic reticulum (ER) anchors, the VAMP-associated proteins (VAPs), by employing bimolecular fluorescence complementation and pull-down set-ups. The ORP–VAP interactions localize frequently at distinct subcellular sites, shown in several cases to represent membrane contact sites (MCSs). Using established ORP ligand-binding domain mutants and pull-down assays with recombinant proteins, we show that ORP liganding regulates the ORP–VAP association, alters the subcellular targeting of ORP–VAP complexes, or modifies organelle morphology. There is distinct protein specificity in the effects of the mutants on subcellular targeting of ORP–VAP complexes. We provide evidence that complexes of human ORP2 and VAPs at ER–lipid droplet interfaces regulate the hydrolysis of triglycerides and lipid droplet turnover. The data suggest evolutionarily conserved, complex ligand-dependent functions of ORP–VAP complexes at MCSs, with implications for cellular lipid homeostasis and signaling.

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Abbreviations

BiFC:

Bimolecular fluorescence complementation

EM:

Electron microscopy

ER:

Endoplasmic reticulum

FFAT:

Two phenylalanines in an acidic tract

GST:

Glutathione-S-transferase

LD:

Lipid droplet

MCS:

Membrane contact site

OHC:

Hydroxycholesterol

ORD:

OSBP-related ligand-binding domain

ORP:

OSBP-related protein

OSBP:

Oxysterol-binding protein

Osh:

OSBP homolog (in yeast)

PI4P:

Phosphatidylinositol-4-phosphate

PIP:

Phosphoinositide

PM:

Plasma membrane

TG:

Triglyceride

VAMP:

Vesicle-associated membrane protein

VAP:

VAMP-associated protein

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Acknowledgments

Liisa Arala, Eeva Jääskeläinen and Riikka Kosonen are thanked for skillful technical assistance, Prof. M. A. De Matteis (Telethon Institute of Genetics and Medicine, Naples, Italy) for the rabbit VAPA and -B antibodies, and Dr. I. Kaverina (Vanderbilt Univ., Nashville, TN, USA) for the mCherry-GalT1(1–81) construct. We thank Eija Jokitalo and the Electron Microscopy Unit (Institute of Biotechnology, Helsinki, Finland) for assistance with the transmission electron microscopy. The study was supported by the Academy of Finland (Grant 257409 to M.W.-B.), the Finnish Concordia Fund (H. K.), the Sigrid Juselius Foundation, the Novo Nordisk Foundation, the Liv ovh Hälsa Foundation, the Finnish Foundation for Cardiovascular Research, and the Magnus Ehrnrooth Foundation (V. M. O.). The authors declare no conflicts of interest.

Author information

Authors and Affiliations

  1. Minerva Foundation Institute for Medical Research, Biomedicum 2U, Tukholmankatu 8, 00290, Helsinki, Finland

    Marion Weber-Boyvat, Henriikka Kentala & Vesa M. Olkkonen

  2. Cell and Molecular Biology Program, Institute of Biotechnology, University of Helsinki, 00014, Helsinki, Finland

    Johan Peränen

  3. Institute of Biomedicine, Anatomy, University of Helsinki, 00014, Helsinki, Finland

    Vesa M. Olkkonen

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  1. Marion Weber-Boyvat
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  2. Henriikka Kentala
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  3. Johan Peränen
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  4. Vesa M. Olkkonen
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Corresponding author

Correspondence to Vesa M. Olkkonen.

Electronic supplementary material

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18_2014_1786_MOESM1_ESM.tif

Supplementary Fig. S1 FFAT motif containing ORPs interact with VAPB at specific sites in the cell. Fluorescence imaging of HuH7 cells expressing different VnORP constructs in combination with VcVAPB and mCherry-VAPB. The BiFC signals indicating physical interaction/proximity of the ORPs and VAPB are shown in the top row, and merge of the BiFC and mCherry-VAPB fluorescence in the bottom row. (TIFF 10164 kb)

18_2014_1786_MOESM2_ESM.tif

Supplementary Fig. S2 Impact of inositol-phosphate-binding cleft mutations (mPIP) on the ORP–VAPA interaction. (A) HuH7 cells expressing the indicated wt or mPIP VnORP2 and VnORP4L constructs were lysed and subjected to GST-VAPA pull-down. Cell lysates and pull-downs were analyzed by Western blotting with anti-GFP antibodies. The GST-VAPA band was visualized as an unspecific band cross-reacting with the secondary antibody. (B) Quantification of three repeats of the experiment in (A). Shown are averages and standard deviations; *p < 0.05. (C and D) Fluorescence imaging of HuH7 cells expressing wt or mutant VnORP constructs [ORP4L (C), ORP2 (D)] in combination with VcVAPA and mCherry-VAPA. BiFC signal intensities were analyzed in a minimum of 100 cells per condition and normalized to the cotransfection marker mCherry-VAPA. Shown are BiFC signal intensity averages and standard deviations. (TIFF 43630 kb)

18_2014_1786_MOESM3_ESM.tif

Supplementary Fig. S3 Electron microscopic analysis of cells expressing the VnORP9L and VcVAPA. (A) Transmission electron microscopy images of HuH7 cells expressing either the empty vectors (control) or VnORP9 in combination with VcVAPA. (B) Quantification of the thickness of ER sheets/tubules in control and ORP9L-VAPA-expressing cells. Shown are averages and SEM of 20 ER samples analyzed per condition; ***p < 0.005. (TIFF 43631 kb)

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Supplementary Fig. S4 Impact of the ORP2 inositol-phosphate-binding cleft mutant (mPIP) on the distribution and total area of lipid droplets (LD). HuH7 cells expressing the indicated wt or mPIP VnORP2 and VcVAPA (BiFC), with the LD (Bodipy-C12) and nuclei (DAPI) co-stained. (A) Fluorescence microscopy images displaying the three channels and their merge. (B) Quantification of total LD area in a minimum of 20 cells per condition. Shown are averages and standard deviations. (C). The LD distribution in ORP2(wt)– and ORP2(mPIP)–VAPA-expressing cells. LD distribution was analyzed in a minimum of 10 cells per condition. The center of the nucleus was used to define the cell center. Shown are averages and SEM. (TIFF 43632 kb)

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Weber-Boyvat, M., Kentala, H., Peränen, J. et al. Ligand-dependent localization and function of ORP–VAP complexes at membrane contact sites. Cell. Mol. Life Sci. 72, 1967–1987 (2015). https://doi.org/10.1007/s00018-014-1786-x

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  • DOI: https://doi.org/10.1007/s00018-014-1786-x

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