© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs212522. doi:10.1242/jcs.212522

RESEARCH ARTICLE The VLDL regulates membrane receptor trafficking and non-genomic signaling Nancy Nader, Maya Dib, Raphael Courjaret, Rawad Hodeify, Raya Machaca, Johannes Graumann and Khaled Machaca*

ABSTRACT the plasma membrane and interact with the classical P4 receptor, is Progesterone mediates its physiological functions through activation of nonetheless effective at mediating non-genomic P4 signaling both transcription-coupled nuclear receptors and seven-pass- (Bandyopadhyay et al., 1998; Dressing et al., 2011; Peluso et al., transmembrane progesterone receptors (mPRs), which transduce 2002). These results argued for the presence of membrane P4 the rapid non-genomic actions of progesterone by coupling to various receptors that are distinct from the nuclear P4 receptors. In 2003, the signaling modules. However, the immediate mechanisms of action Thomas laboratory identified a family of membrane progesterone downstream of mPRs remain in question. Herein, we use an untargeted receptors (mPRs) from fish ovaries (Zhu et al., 2003a,b) that belong quantitative proteomics approach to identify mPR interactors to better to the progestin and adiponectin (AdipoQ) receptor family (also define progesterone non-genomic signaling. Surprisingly, we identify named PAQ receptors). However, the signal transduction cascade the very-low-density receptor (VLDLR) as an mPRβ downstream of mPRs that mediates the non-genomic actions of P4 (PAQR8) partner that is required for mPRβ plasma membrane remains unclear. localization. Knocking down VLDLR abolishes non-genomic The non-genomic action of mPR and the ensuing signaling progesterone signaling, which is rescued by overexpressing VLDLR. cascade have been studied extensively over the years in frog and fish Mechanistically, we show that VLDLR is required for mPR trafficking oocytes, where progesterone releases oocyte meiotic arrest. from the endoplasmic reticulum to the Golgi. Taken together, our data Vertebrate oocytes must undergo a maturation period before they define a novel function for the VLDLR as a trafficking chaperone become fertilization competent and are able to support embryonic required for the mPR subcellular localization and, as such, non- development. Oocyte maturation encompasses progression through genomic progesterone-dependent signaling. meiosis and arrest at metaphase II until fertilization (Machaca, 2007). It is defined by drastic cellular remodeling characterized by This article has an associated First Person interview with the first author the dissolution of the nuclear envelope (referred to as germinal of the paper. vesicle breakdown; GVBD), extrusion of the first polar body, condensation and arrest in metaphase of meiosis II KEY WORDS: Membrane , VLDLR, Meiotic (Bement and Capco, 1990; Nader et al., 2013; Sadler and Maller, arrest, Oocyte, Trafficking, Oocyte maturation, Endoplasmic 1985; Smith, 1989; Voronina and Wessel, 2003). Prior to oocyte reticulum, Golgi, Progesterone maturation and for prolonged periods of time, fully grown vertebrate oocytes are arrested at prophase of meiosis I, as they grow and stock INTRODUCTION molecular components essential for future development (Smith, Progesterone (P4) is a steroid hormone that regulates various 1989; Voronina and Wessel, 2003). P4 releases Xenopus oocyte reproductive processes in females, including ovulation, meiotic arrest by ultimately activating maturation-promoting factor implantation and sexual differentiation. Signaling downstream of (MPF; the cyclin-B–cdc2 complex), the key driver of meiosis P4 has been studied primarily through the activation of nuclear (Nader et al., 2013; Nebreda and Ferby, 2000). In amphibian receptors that act as transcription factors to stimulate P4-dependent oocytes, treatment with the membrane-impermeant P4 conjugated expression (Ellmann et al., 2009). However, P4 is also known to BSA (P4–BSA), efficiently releases meiotic arrest. These and to transduce rapid non-genomic signals that link to cAMP, Ca2+ and other results indicate that P4 acts through a membrane receptor the mitogen-activated kinase (MAPK) cascade, among other rather than the classical nuclear receptor (Bandyopadhyay et al., signaling modules (Valadez-Cosmes et al., 2016). Initially, 1998; Blondeau and Baulieu, 1984; Josefsberg Ben-Yehoshua activation of these signaling cascades was attributed to the et al., 2007), although there is evidence for a potential role for the classical ability of the progesterone receptor to interact with nuclear receptor as well (Bayaa et al., 2000; Tian et al., 2000). There cytoplasmic factors (Dressing et al., 2011). However, non- is also evidence that other steroids release frog oocyte meiotic arrest, genomic actions of progestins have been demonstrated in several with testosterone being the physiological inducer of oocyte tissues that do not express classical progesterone receptors, and P4 maturation in vivo (Lutz et al., 2001). In 2007, Ben-Yehoshua coupled to bovine serum albumin (BSA), which is unable to cross et al. reported the identification of a Xenopus laevis mPR ortholog, mPRβ (also known as PAQR8, herein referred to as mPR for simplicity) as the receptor functionally responsible for the Department of Physiology and Biophysics, Weill Cornell Medicine Qatar, Education City – Qatar Foundation, P.O. Box 24144, Doha, Qatar. resumption of meiosis in response to P4 (Josefsberg Ben- Yehoshua et al., 2007). It is well documented that high levels of *Author for correspondence ([email protected]) cAMP and protein kinase A (PKA) are important in maintaining K.M., 0000-0001-6215-2411 meiotic arrest in vertebrates (Bravo et al., 1978; Cho et al., 1974; Conti et al., 2002; Gallo et al., 1995; Lutz et al., 2000; Maller and

Received 25 October 2017; Accepted 17 April 2018 Krebs, 1977; Meijer and Zarutskie, 1987; Sadler et al., 2008; Sheng Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212522. doi:10.1242/jcs.212522 et al., 2001; Stern and Wassarman, 1974). In Xenopus oocytes, the We then checked the trafficking of the two constructs GFP–mPR high levels of cAMP–PKA are maintained, at least in part, through and mPR–GFP by performing confocal imaging of live oocytes. To the action of the constitutively active Gαs-coupled G protein- mark the plasma membrane, we also expressed the resident plasma coupled receptor GPR185 (Ríos-Cardona et al., 2008), the homolog membrane (PM) protein TMEM–mCherry, which encodes a Ca2+- of mammalian GPR3, which has been found to block oocyte activated Cl− channel (Yu et al., 2010). A z-stack of confocal images maturation in mouse oocytes (Freudzon et al., 2005; Mehlmann was acquired across the oocyte (Fig. S1), with the orthogonal et al., 2002, 2004; Norris et al., 2007). mPR activation does not section through this stack representing the subcellular distribution of seem to signal through Gαi to inhibit adenylate cyclase since the as previously described (Yu et al., 2010). We have treatment with pertussis toxin was ineffective at blocking P4- previously shown that TMEM–mCherry targets to the PM and that induced oocyte maturation (Mulner et al., 1985; Olate et al., 1984; its membrane residence is not affected throughout oocyte Sadler et al., 1984). Similarly, maturation in mouse oocytes also maturation (Nader et al., 2014; Yu et al., 2010). Interestingly, the seems to be independent from the action of the Gi subunit GFP–mPR construct, which is unable to release meiotic arrest, was (Mehlmann et al., 2006). Moreover, although high levels of mostly intracellular and unable to localize to the PM (Fig. 1E; Fig. cAMP are essential in maintaining oocyte meiotic arrest, oocyte S1B), whereas a significant proportion of the functional mPR–GFP maturation progresses without any changes in cAMP or PKA levels localized to the PM (microvilli) in addition to localizing (Nader et al., 2016). These findings argue for a positive signal intracellularly (Fig. 1E; Fig. S1A). downstream of mPR that overrides the cAMP–PKA inhibitory GFP–mPR- and mPR–GFP-expressing oocytes were stained with signal (Nader et al., 2016). This positive signal is likely to be wheat germ agglutinin (WGA) to label the plasma membrane, initiated through the P4–mPR axis to release the oocyte from allowing for the quantification of the distribution of mPR at the PM meiotic arrest. (Courjaret et al., 2016; El-Jouni et al., 2008). mPR–GFP was found Here, we employ an untargeted proteomics approach to identify to be significantly enriched at the PM (38.91±1.89%, mean±s.e.m.; the mPR interactome to better define signaling downstream of n=10), whereas GFP–mPR was mainly restricted to the cytosol, mPR. We identify the very-low-density lipoprotein receptor with only 16.18±0.98% (n=12) at the PM (Fig. 1F). The (VLDLR) as an mPR-interacting protein, and show that VLDLR intracellular component of mPR–GFP could represent the is essential for mPR plasma membrane localization, and, as such, proportion of the receptor that is undergoing biogenesis, and/or its signaling function. VLDLR acts as a molecular chaperone that saturation of the trafficking machinery when mPR is overexpressed. is required for the trafficking of mPR from the endoplasmic Furthermore, our conservative quantification approach using the reticulum (ER) to the Golgi. In the absence of VLDLR, mPR peak of WGA staining as defining the PM (see Materials and concentrates in the ER and does not reach the cell membrane where Methods), is likely to underestimate the intracellular proportion of it performs its signaling function. Hence, the VLDLR plays an both GFP-tagged mPRs given the opacity and size of the oocyte. essential role in regulating mPR trafficking and signaling during Nevertheless, the subcellular localization data clearly show that oocyte maturation. GFP–mPR is defective in its trafficking to the PM, which correlates with its inability to accelerate oocyte maturation when RESULTS overexpressed (Fig. 1A–D). This argues that mPR exerts its GFP-tagged mPR – functional analysis and localization signaling function only when it localizes to the PM and is unable To characterize the subcellular localization of the Xenopus mPRβ to signal when localizing intracellularly. Furthermore, it shows that (mPR) in the absence of good anti-mPR antibodies, we tagged mPR mPR does not signal constitutively when overexpressed and still with GFP at its N- (GFP–mPR) or C-terminus (mPR–GFP). In order requires P4. to test the functionality of the GFP-tagged mPR, the physiological effects of overexpressed untagged mPR, GFP–mPR and mPR–GFP Identification of VLDLR as an mPR-interacting protein were tested on progesterone (P4)-induced oocyte maturation as The GFP–mPR and mPR–GFP constructs provide perfect tools to compared to the expression of GFP alone (Fig. 1). In accordance define mPR-interacting proteins that mediate its non-genomic with a previous report (Josefsberg Ben-Yehoshua et al., 2007), we actions. Building on the fact that GFP–mPR is not functional, found that overexpressing wild-type mPR significantly (P=0.045) whereas mPR–GFP mediates oocyte maturation with a similar potentiated oocyte maturation at suboptimal concentrations of P4 efficiency to that of the untagged receptor, we used a quantitative concentration (3×10−8 M) (Fig. 1A). Similarly, the C-terminally unbiased proteomics approach to identify proteins that preferentially tagged mPR, mPR–GFP was able to potentiate oocyte maturation at interact with the functional mPR–GFP (Fig. 2A). Proteins that suboptimal P4 concentrations (Fig. 1A, P=0.035). In contrast, no interact preferentially with mPR–GFP as compared to the non- potentiation of oocyte maturation was observed when GFP alone or functional GFP–mPR are likely to be important for its trafficking the N-terminally tagged GFP–mPR proteins were overexpressed and/or signaling functions. Proteomics approaches are notorious for (Fig. 1A). Although the maximum GVBD percentage was not identifying potential non-specific proteins given the biochemical significantly affected by expression of the different mPR constructs approaches used and the dynamic range of the mass spectrometry when an optimal concentration of 3×10−7 M P4 was used (Fig. 1B), (MS) approach itself. To minimize these potential artifacts, we oocytes overexpressing mPR and mPR–GFP consistently reached pulled down GFP–mPR or mPR–GFP from oocytes overexpressing 50% GVBD around 3 h faster (P=0.048 and P=0.022, respectively) either construct using anti-GFP-linked beads (Fig. 2A). This avoids when compared to oocytes overexpressing GFP alone or GFP–mPR contamination with the endogenous mPR and isolates the (Fig. 1C,D). Both GFP–mPR and mPR–GFP were expressed at interactome of the specific overexpressed protein. Both constructs similar levels (Figs 1E and 2B). These results show that the C- were expressed to similar levels and were pulled-down efficiently terminally tagged mPR is functional and replicates the activity of the (Fig. 2B), followed by trypsin digestion and dimethyl labeled with wild-type protein. In contrast, tagging mPR at its N-terminus heavy (mPR–GFP) or light (GFP–mPR) isotopes before MS somehow interferes with its function, resulting in a functionally analysis as outlined in Fig. 2A. The ‘heavy-over-light’ ratio defective protein. identified relatively few candidates that preferentially bind the Journal of Cell Science

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Fig. 1. Functional characterization of GFP- tagged mPR. (A,B) Effect of mPR on oocyte maturation. Oocytes were injected with RNA coding for GFP, wild-type mPR (untagged), mPR–GFP (C-terminally tagged) or GFP– mPR (N-terminally tagged), and after 48 h, were treated with progesterone (P4) overnight at suboptimal (3×10−8 M) (A), or optimal (3×10−7 M) (B) concentrations. Oocyte maturation was scored ∼16 h after P4 treatment by the appearance of a white spot on the oocyte animal hemisphere, which is indicative of germinal vesicle breakdown (GVBD). (C) Time needed for 50% of the oocytes to reach GVBD after adding P4 (3×10−7 M) in the presence of overexpressed GFP, mPR, mPR–GFP or GFP–mPR as indicated. (D) Representative GVBD time courses in response to P4 (3×10−7 M) in oocytes overexpressing GFP, mPR, mPR– GFP or GFP–mPR as indicated. The data are normalized to the values in GFP-injected cells and a non-linear curve was fitted to the data. (E) Representative orthogonal sections from a confocal stack of images (see Fig. S1) taken 48 h after injecting RNAs encoding mPR–GFP or GFP–mPR along with the PM marker, TMEM–mCherry. Scale bar: 2 µm. (F) Histogram showing the percentage of mPR–GFP or GFP–mPR at the PM. Quantitative results in A–C and F are mean±s.e.m. for three or more experiments. *P<0.05; ***P<0.001.

functional mPR–GFP as compared to GFP–mPR (Fig. 2C; Role of VLDLR in P4–mPR-induced maturation Table S1). VLDLR emerged as the most consistently and highly To directly test the role of VLDLR in P4–mPR signaling, we enriched protein (Fig. 2C; Table S1). We therefore focused on the knocked down VLDLR expression and tested the effects on oocyte VLDLR as a potential mediator of P4-dependent signaling through maturation. Antisense oligonucleotides were effective at knocking mPR to drive the oocyte maturation and meiosis progression. down VLDLR RNA levels as compared to the control sense To further characterize the role of VLDLR, we engineered N- and oligonucleotides (Fig. 3A). Although the sense oligonucleotides C-terminally mCherry-tagged VLDLR constructs. In order to resulted in some decrease in VLDLR RNA levels as compared to validate the quantitative proteomics results and confirm the that found in control uninjected oocytes, the antisense association of VLDLR with the C-terminally tagged mPR–GFP, oligonucleotides almost completely abrogated VLDLR RNA we co-expressed VLDLR tagged with mCherry at either the N- (Ch– (Fig. 3A). The antisense effect was specific to VLDLR as no VLDLR) or the C-terminus (VLDLR–Ch) with mPR–GFP and significant changes in the levels of mPR RNA were detected performed an immunoprecipitation using anti-GFP beads (Fig. 2D). (Fig. 3A). The antisense oligonucleotides were also effective at Immunoprecipitation of mPR–GFP pulled down VLDLR–Ch and blocking exogenous VLDLR–Ch protein expression (Fig. 3B). Ch–VDLDR, indicating that the two proteins are part of the same Interestingly, knocking down VLDLR expression strongly and complex in situ (Fig. 2D). significantly (P<0.0001) inhibited P4-induced oocyte maturation VLDLR is part of the low-density-lipoprotein (LDL) (Fig. 3C). The inability of oocytes to mature following VLDLR transmembrane receptor family that localizes to the PM (Go and knockdown was confirmed biochemically by assaying the Mani, 2012). Consistent with this, the Xenopus VLDLR tagged at activation of both MAPK (ERK1/2) and MPF, two kinases known either end localized to the PM, as indicated by the WGA co-staining to be activated downstream of P4 treatment and are required for (Fig. 2E). Although both VLDLR constructs primarily localize to oocyte maturation (Fig. 3D). The MAPK cascade is activated the PM (Fig. 2E), we observed a modest but significant (P=0.012) downstream of progesterone and contributes to the induction of enrichment of the N-terminally tagged Ch–VLDLR (67.31± MPF (cyclin B–Cdc2) the master regulator of oocyte maturation 1.876%; mean±s.e.m.; n=29) at the PM as compared to VLDLR– (Castro et al., 2001; Palmer and Nebreda, 2000). MAPK activation Ch (58.47±2.837%; n=29) (Fig. 2F). is detected by phosphorylation, whereas MPF activation is followed Journal of Cell Science

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Fig. 2. Identification of VLDLR as an mPR- interacting protein. (A) Illustration of the experimental design to identify proteins that selectively interact with the functional mPR– GFP construct. (B) Oocytes were injected with mPR–GFP or GFP–mPR RNAs and, 48 h later, lysates immunoprecipitated with anti-GFP magnetic microbeads. Whole-cell lysates (input) and eluates from the anti-GFP beads (IP) were examined by western blotting using an anti-GFP antibody. (C) Plot of the heavy:light ratios (mPR–GFP/GFP–mPR) from three separate mass spectrometry experiments showing the consistent enrichment of VLDLR in its selective interaction with mPR–GFP. (D) Oocytes were either uninjected (Naïve) or injected with RNA coding for the C-terminally tagged mPR–GFP alone (injected) or co- injected with either the N- (Ch–VLDLR) or C-terminally (VLDLR–Ch) mCherry-tagged VLDLR and allowed to express proteins for 48 h. After cross-linking, mPR–GFP was immunoprecipitated using anti-GFP magnetic microbeads. Fractions from the whole lysates (input) and the GFP-binding eluate (IP) were examined by western blotting using anti-GFP and anti-mCherry/anti-RFP antibodies. (E) Representative orthogonal sections of an oocyte stained with WGA and overexpressing VLDLR–mCherry or mCherry–VLDLR as indicated. Scale bar: 2 µm. (F) Histogram showing the percentage of VLDLR–mCherry or mCherry–VLDLR at the PM (mean±s.e.m. of 29 oocytes/condition). *P<0.05.

by dephosphorylation of the kinase subunit Cdc2 (Fig. 3D). Tubulin hints to a role for VLDLR in regulating mPR trafficking. This would was used as a loading control. be functionally critical since the GFP–mPR, which is unable to reach To confirm that the antisense effect is specific to the knockdown the PM does not support oocyte maturation. To test whether VLDLR of the VLDLR and not due to some offsite effects, we tested modulates mPR trafficking, we used a progesterone analog coupled whether overexpression of the VLDLR can rescue the VLDLR to BSA and fluorescein (that is membrane impermeant because of knockdown. Overexpressing untagged VLDLR (10 ng/oocyte) the BSA moiety) that allows easy imaging and quantification of completely reversed the inhibition of oocyte maturation mediated endogenous mPR at the PM. To validate P4–BSA–fluorescein as a by VLDLR knockdown (Fig. 3E), confirming the specificity of reliable reagent to quantify mPR at the PM, we overexpressed mPR, VLDLR knockdown on P4-mediated maturation. In addition, which resulted in a significant (P=0.0042) increase in P4–BSA– untagged mPR overexpression (10 ng/oocyte) was also able to fluorescein binding (Fig. 4A,B). In contrast, knocking down significantly rescue the inhibition of oocyte maturation mediated by VLDLR expression (antisense) results in a significant (P<0.0001) VLDLR knockdown, from 17% of oocytes showing GVBD to 57% 60% decrease of endogenous mPR localizing to the plasma (Fig. 3E). Interestingly, the rescue of oocyte maturation was coupled membrane as compared to the sense control or naïve untreated to a significant increase of mPR PM levels, as discussed below oocytes (Fig. 4A,B). These data show that VLDLR is required for (Fig. 4F). These data collectively show that VLDLR is required for the trafficking of endogenous mPR to the PM. oocyte maturation and to mediate the activation of the signaling To better define the mPR trafficking defect in the absence of cascade downstream of P4-mPR. VLDLR, we tested the effect of VLDLR knockdown on trafficking of overexpressed mPR. Oocytes were co-injected with RNAs VLDLR is essential for mPR PM localization that express mPR–GFP and TMEM–mCherry to mark the PM along The high enrichment of VLDLR within the functional mPR–GFP with VLDLR sense or antisense oligonucleotides (Fig. 4C–E). complex that traffics normally to the plasma membrane, as The co-injection of mPR-encoding RNA with antisense compared to the defective GFP–mPR that localizes intracellularly, oligonucleotides targeting VLDLR was designed to knockdown Journal of Cell Science

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Fig. 3. VLDLR is required for the release of oocyte meiotic arrest. (A) Knockdown of VLDLR expression. Oocytes were injected with VLDLR sense oligonucleotides, as a control, or the corresponding antisense oligonucleotides to knockdown VLDLR expression. RNA was prepared 48 h later and analyzed by qRT-PCR to determine the efficacy of the knockdown on VLDLR and mPR expression. Data are expressed as relative levels of VLDLR and mPR mRNA transcripts after normalizing to the levels of Xenopus ornithine decarboxylase (xODC) mRNA. Naïve, uninjected oocytes. (B) Naïve or VLDLR–mCherry- overexpressing oocytes were injected with VLDLR sense or antisense oligonucleotides and cell extracts were analyzed by western blotting using anti-mCherry antibodies 48 h later. Tubulin is shown as a loading control. (C) VLDLR is required for P4-dependent oocyte maturation. Oocytes were injected with VLDLR sense or antisense oligonucleotides and, 48 h later, incubated in P4-containing solution overnight. The percentage of oocytes that had undergone GVBD normalized to the naïve treatment is shown. (D) Western blot assessing the MAPK ERK1/2 and Cdc2 phosphorylation state for the different treatments as indicated. Ooc. refers to immature oocytes before progesterone treatment and Egg to mature eggs. Tubulin is shown as a loading control. (E) VLDLR knockdown rescue. Oocytes were injected with VLDLR sense or antisense oligonucleotides in the presence or absence of untagged VLDLR (10 ng RNA/oocyte) or untagged mPR (10 ng RNA/oocyte) as indicated. P4 was added 48 h later and the percentage of oocytes undergoing GVBD was normalized to the GVDB recorded with VLDLR sense-injected oocytes. Quantitative results are mean±s.e.m. for three or more experiments. *P<0.05; **P<0.01; ***P<0.001.

VLDLR expression at the same time as mPR was overexpressed specific to mPR. The fact that VLDLR interacts with mPR would over a 48 h timecourse to address the role of the VLDLR in the argue for a specific effect. However, to directly test whether VLDLR biogenesis and PM targeting of mPR. We found that VLDLR is a specific chaperone of mPR, we measured the effect of VLDLR knockdown inhibits the trafficking of mPR–GFP to the PM and its knockdown on total PM surface area by determining the membrane colocalization with TMEM–mCherry as observed in the orthogonal capacitance. Knockdown of VLDLR had no significant effect confocal sections (Fig. 4C). This was also apparent from the shift of on membrane capacitance, which measured 248+11.3, 243+ 9.7 mPR–GFP fluorescence towards the interior of the cell following and 231+8.2 nF (n=12 oocytes/condition, mean±s.e.m.) in naïve, VLDLR knockdown (Fig. 4D). Quantification of overexpressed VLDLR sense and VLDLR antisense-injected oocytes respectively. mPR PM residence in oocytes where VLDLR has been knocked We further tested whether VLDLR affects the trafficking of Orai1, a down compared to the amount in the sense control, reveals a PM Ca2+ channel that is required for store-operated Ca2+ entry significant (P<0.0001) decrease, by ∼20%, in the amount of mPR (SOCE) and that we have previously shown recycles at the PM at the PM (Fig. 4E). The fact that VLDLR knockdown results in a (Yu et al., 2010). Again, VLDLR knockdown had no significant more substantial decrease (60%) in endogenous mPR at the PM than effect on the SOCE current, arguing that it does not affect Orai1 what is seen when mPR is overexpressed (20% decrease) argues for trafficking (Fig. 4G). These results rule out a general effect of a saturation effect of the trafficking machinery with excess mPR, VLDLR on plasma membrane recycling and support a specific role thus allowing mPR to reach the PM even when VLDLR is limiting. for the VLDLR in regulating mPR trafficking to the PM. This is consistent with the rescue of oocyte maturation observed in antisense VLDLR-treated oocytes (Fig. 3D). To directly test this Colocalization of mPR and VLDLR conclusion, we overexpressed mPR in oocytes where VLDLR has VLDLR is a type 1 single-pass transmembrane protein with an been knocked down (Fig. 4F). This resulted in a partial rescue of the extracellular N-terminus and cytosolic C-terminus. To further ability of mPR to localize to the PM. Hence, excess mPR saturates characterize the mPR–VLDLR interaction, we co-expressed and the VLDLR-dependent trafficking regulation. imaged the different combinations of N- and C-terminally tagged Although our data show a clear role for the VLDLR in regulating mPR and VLDLR (Fig. 5). Co-expression of the functional the trafficking of mPR, it is not clear whether this effect is due to a C-terminally tagged mPR–GFP with VLDLR tagged at either broad deregulation of the trafficking machinery or whether it is end, shows colocalization of both proteins at the PM (Fig. 5A,B). Journal of Cell Science

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Fig. 4. VLDLR is essential for mPR localization to the plasma. (A,B) Effect of VLDLR knockdown on endogenous mPR trafficking. Oocytes were left untreated (Naïve) or injected with mPR RNA or VLDLR sense or antisense oligonucleotides and stained with P4–BSA–Fluorescein 48 h later to quantify the levels of endogenous mPR at the plasma membrane. (A) Low-magnification confocal images showing P4–BSA–Fluorescein staining for the different treatments as indicated. The control treatment shows background staining in the absence of P4–BSA–FITC. Scale bar: 50 µm. (B) Quantification of the P4–BSA–FITC staining from ImageJ in the different treatments normalized to the average in control uninjected oocytes (Naïve). (C–E) Effect of VLDLR knockdown on trafficking of overexpressed mPR. Oocytes were co-injected with RNAs expressing mPR and the PM marker TMEM–mCherry in the presence of VLDLR sense or antisense oligonucleotides. Confocal z-stacks were acquired 48 h later with the pinhole at 1 airy unit. ImageJ was used to quantify the fluorescence in a specific ROI. (C) Representative orthogonal sections of the two individual oocytes highlighted in green in E. Scale bar: 2 µm. (D) GFP and mCherry fluorescence intensities along the z-stack section from the two individual oocytes highlighted in green in E. (E) Quantification of the percentage of overexpressed mPR–GFP localized at the PM following injection of VLDLR sense or antisense oligonucleotides. Data were normalized to the average of mPR percentage at the PM from VLDLR sense-injected oocytes. (F) VLDLR-knockdown rescue experiment. Oocytes were uninjected (Naïve) or injected with VLDLR antisense oligonucleotides with or without mPR RNA. Endogenous mPR at the PM was quantified 48 h later through P4–BSA–FITC staining. P4–BSA–FITC fluorescence in a specific ROI was quantified using ImageJ and the data normalized to the average P4–BSA–FITC fluorescence from naïve oocytes. (G) Oocytes were either uninjected (Naïve) or injected with VLDLR sense or antisense oligonucleotides as indicated, and the endogenous Ca2+-activated Cl currents, as a measure of SOCE, were recorded 48 h later as described in the Materials and Methods section. Quantitative results are mean±s.e.m. for three or more experiments or as indicated by individual data points. **P<0.01; ***P<0.001.

Furthermore, mPR–GFP colocalizes intracellularly with VLDLR– slightly but significantly lowered the amount of endogenous and Ch, which shows a partial intracellular distribution (Fig. 5B). overexpressed mPR at the plasma membrane (Fig. 5F,G). In Ch–VLDLR is almost exclusively at the PM (Fig. 5A), whereas contrast, overexpressing Ch–VLDLR significantly increased the VLDLR–Ch can be clearly found intracellularly (Fig. 5B). proportion of endogenous mPR at the plasma membrane, whereas By contrast, GFP–mPR does not colocalize with either VLDLR no effect was found on overexpressed mPR, probably due to the construct and exhibits a reticular intracellular distribution saturation of the exocytosis machinery (Fig. 5F,G). These results reminiscent of the ER (Fig. 5C,D). These results show that when show that when the VLDLR is able to interact with mPR this alters mPR is tagged with GFP at its N-terminus it is unable to interact its membrane residence and trafficking. Collectively, our data with VLDLR. This was confirmed by immunoprecipitation; pulling validate VLDLR as a novel trafficking chaperone of mPR, and down GFP–mPR did not co-immunoprecipitate VLDLR (Fig. 5E). importantly, in that capacity, as a critical physiological regulator of Remarkably, overexpressing VLDLR–Ch, which localizes oocyte maturation. intracellularly to a higher degree than Ch–VLDLR (Fig. 2E,F), Journal of Cell Science

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Fig. 5. VLDLR is required for mPR trafficking. (A,B) Representative focal plane images of WGA-stained oocytes expressing mPR–GFP with mCherry–VLDLR (A) or VLDLR–mCherry (B). (C) Representative intracellular focal plane and orthogonal sections of oocytes overexpressing GFP–mPR with VLDLR tagged at its N- or C-terminus. The typical reticular ER structure surrounding pigment granules, which are indicated by stars in panels A–C, and the VLDLR–Ch- or mPR–GFP- positive puncta representative of the Golgi are indicated by arrowheads. Scale bars: 2 µm. (D) Higher resolution view of the box indicated in the merge image in C to better highlight the VLDLR-positive Golgi structures and the reticular ER appearance indicated by the GFP–mPR staining. A cartoon rendering showing the ER and Golgi (labeled G) is shown in the bottom-right image. (E) Lack of physical interaction between the N-terminally tagged GFP–mPR and mCherry-tagged VLDLR. Oocytes were injected with RNA coding for GFP–mPR along with N- (Ch–VDLDR) or C-terminally (VLDLR–Ch) tagged VLDLR and allowed to express for 48 h. This was followed by crosslinking, lysing and immunoprecipitation using anti-GFP magnetic microbeads. Fractions from the whole lysates (input) and eluates from the anti-GFP beads (IP) were examined by western blotting using anti-GFP, anti-mCherry and anti-RFP antibodies. (F) Quantification of endogenous mPR PM residence in the presence of overexpressed VLDLR. Oocytes were injected with VLDLR–Ch or Ch–VLDLR RNA and stained with P4–BSA–FITC 48 h later. Data were normalized to the average P4–BSA–FITC fluorescence in naïve (uninjected) oocytes. (G) Quantification of mPR-GFP at the PM following expression of mPR-GFP alone or with mCherry-VLDLR or VLDLR-mCherry as indicated. Oocytes were stained with WGA and confocal z-stacks taken 48 h later. Quantitative results are mean±s.e.m. for three or more experiments or as indicated by individual data points. ***P<0.001.

VLDLR is required for mPR trafficking from the ER to the Golgi the cortex (Colman et al., 1985). As is the case in mammalian cells, As shown in Fig. 5A,B, the intracellular distribution of mPR–GFP the Golgi stacks fragment during M-phase in the oocyte (Colman exhibits the typical reticular ER distribution (stars), in addition to a et al., 1985). To determine whether the mPR-positive structures are portion of the protein distributing to punctate structures that are indeed Golgi complexes, we expressed the Golgi-resident enzyme reminiscent of the Golgi complex in the oocyte (arrowheads). In N-acetylgalactosaminyltransferase-2 (GalNac) fused to GFP contrast to the tight reticular distribution typical of the ER in the frog (Storrie et al., 1998). To differentiate the Golgi complexes from oocyte (Figs 5 and 6), expression of GFP or mCherry alone shows a the ER we co-expressed GalNac–GFP (Golgi) with an ER marker diffuse cytoplasmic distribution with exclusion from the areas (KDEL–mCherry) (Fig. 6A). KDEL–Cherry shows the typical containing pigment granules (Fig. S2A). Although, as far as we are reticular ER distribution in the oocyte, whereas the Golgi marker aware, the Golgi has not been visualized in Xenopus oocytes using GalNac–GFP distributes in distinct punctate structures (Fig. 6A, fluorescence probes, it was studied by electron microscopy in the arrowheads) that do not overlap with the ER (Fig. 6A, top row). 1980s. The oocyte, given its large size, has multiple Golgi Furthermore, and consistent with the literature (Colman et al., complexes that are distributed both deep in the cytosol and within 1985), the Golgi complexes fragmented during meiosis as Journal of Cell Science

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Fig. 6. VLDLR is essential for mPR trafficking from the ER to the Golgi. (A) Representative intracellular focal images of individual oocytes co-injected with the Golgi marker GalNac–GFP and the ER marker KDEL–mCherry, or GalNac–GFP and VLDLR–Ch, or mPR–GFP and VLDLR–Ch as indicated. The arrowheads point to representative Golgi puncta. (B) Effect of VLDLR knockdown on mPR–GFP trafficking from the ER to the Golgi. Oocytes were co-injected with RNAs coding for mPR–GFP and the ER marker KDEL–mCherry, with VLDLR sense or antisense oligonucleotides. Confocal z-stacks were acquired 48 h later. The mPR-positive puncta represent the Golgi. Scale bars: 2 µm. (C) Quantification of the number of mPR–GFP-positive Golgi following injection of VLDLR sense or antisense oligonucleotides from images similar to the one in B. (D) Quantification of the number of Golgi (GalNac-GFP positive) in sense and antisense VLDLR- injected oocytes from images similar to those shown in A. Quantitative results are mean±s.e.m. for three or more experiments. ***P<0.001; ns, not significant. illustrated by the dramatic decrease in the GalNac-positive structure top row). In contrast, injecting oocytes with VLDLR antisense in the mature egg (Fig. S2B). Co-expression of VLDLR with the oligonucleotides resulted in a significant reduction of the Golgi marker GalNac–GFP shows that VLDLR–Ch localized to the localization of mPR to the Golgi puncta and a more pronounced Golgi (Fig. 6A, middle row, arrowheads). Therefore, VLDLR–Ch, ER localization (Fig. 6B, bottom row). Quantification of the number in addition to its PM distribution (Fig. 5B), localizes to the Golgi of mPR–GFP-positive Golgi puncta in the sense versus antisense intracellularly. The co-expression of mPR–GFP with VLDLR–Ch VLDLR oligonucleotide-treated oocytes shows a significant shows that mPR–GFP also localizes to the intracellular punctate reduction in the antisense VLDLR oocytes (7.97±1.06 Golgi structures (Fig. 6A, bottom row, arrowheads) identified in the puncta/100 µm2) as compared to their sense-injected counterparts GalNac–GFP/VLDLR–Ch co-expression experiment as Golgi (13.25±1.22 Golgi puncta/100 µm2) (Fig. 6C). To rule out the (Fig. 6A, middle row). These results show that mPR–GFP possibility that VLDLR knockdown has any effect on the Golgi colocalizes to the Golgi with VLDLR–Ch during its biogenesis. directly, we quantified the number of Golgi puncta (GalNac–GFP Interestingly, the N-terminally tagged GFP–mPR does not positive) in sense and antisense VLDLR-injected oocytes, and show colocalize with VLDLR–Ch at the Golgi and is instead enriched no difference in the number of Golgi puncta (Fig. 6D). This shows in the ER (Fig. 5C,D). GFP–mPR does not traffic to the plasma that the VLDLR is not required for Golgi stability but rather for the membrane (Fig. 5C), and does not interact with VLDLR in co- trafficking of mPR from the ER to the Golgi. Therefore, the immunoprecipitation experiments (Fig. 5E). Furthermore, GFP– decreased PM residence of mPR in the absence of the VLDLR is mPR is not functional in terms of signaling downstream of P4 in due to a defect in the transit of mPR from the ER to the Golgi. terms of releasing meiotic arrest (Fig. 1A), consistent with its inability to traffic efficiently to the PM (Fig. 1E). These results DISCUSSION argue that GFP–mPR, given its inability to interact with VLDLR, is The fast non-genomic progesterone signaling via membrane unable to traffic from the ER to the Golgi in transit to the PM. To progesterone receptors has emerged as an important regulator of directly test whether this is the case, we knocked down endogenous biological function in the nervous system, female and male VLDLR expression using antisense oligonucleotides and quantified reproductive tissues, and immune and cancer cells (Dosiou et al., the localization of mPR to the Golgi puncta. If VLDLR is indeed 2008; Dressing et al., 2011; Moussatche and Lyons, 2012; Valadez- required for mPR trafficking from the ER to the Golgi, we would Cosmes et al., 2016). The first mPR was cloned from fish oocytes, expect a decreased Golgi localization of expressed mPR–GFP when followed by investigation for its role in oocyte maturation in endogenous VLDLR is knocked down. This is indeed what we multiple non-mammalian species (Josefsberg Ben-Yehoshua et al., observe. Injecting oocytes with the VLDLR sense oligonucleotides 2007; Thomas et al., 2002; Zhu et al., 2003b). Since then, and given gave the typical punctate mPR-positive Golgi distribution (Fig. 6B, the broad expression profile for mPRs in different tissues in Journal of Cell Science

8 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212522. doi:10.1242/jcs.212522 vertebrates, studies have elucidated important signaling roles for Golgi and then to the PM. Since mPR non-genomic signaling to this gene family in diverse physiological and pathological processes release meiotic arrest requires its membrane residence, the VLDLR (Dressing et al., 2011), including sperm motility (Thomas et al., becomes essential for mPR-dependent signaling, as it is required for 2009; Valadez-Cosmes et al., 2016), female reproduction (Qiu et al., the trafficking and membrane residence of mPR. Knockdown of 2008; Nutu et al., 2007, 2009) and immune cell function (Dressing VLDLR phenocopies mPR inhibition in the oocyte and blocks P4- et al., 2011). dependent release of oocyte meiotic arrest. The VLDLR is a transmembrane lipoprotein receptor of the low- In conclusion, by using an unbiased quantitative proteomics density-lipoprotein receptor (LDLR) family, which consists of approach, we identify the VLDLR as an mPR-interacting protein. seven structurally closely related proteins (May et al., 2005). The We further show that the VLDLR is required for the trafficking and LDLR family is composed of constitutively recycling cell surface PM localization of mPR by mediating its transit from the ER to the receptors that are responsible for the uptake of and other Golgi. These results extend the known functions of this versatile ligands via and lysosomal delivery, primarily in the receptor, to include a chaperone trafficking function for mPR. Given (Gotthardt et al., 2000; May et al., 2005). However, some the broad tissue distribution and co-expression of VLDLR and members of the family, particularly the VLDLR, also mediate mPRs, this raises the intriguing prospect that the VLDLR modulates intracellular signaling cascades especially in the brain (May et al., P4-dependent non-genomic signaling in various physiological and 2005; Ranaivoson et al., 2016). The VLDLR amino acid sequence is pathological conditions. It is, as such, important to further explore highly conserved during evolution with ∼95% identity between the role of VLDLR in mPR-dependent non-genomic signaling, mammals, and more than 75–80% identity among vertebrates especially given the role of mPRs in cancer, reproductive regulation (humans, chickens and frogs), implying an essential conserved in both genders, and sugar . function in these species (Bujo and Yamamoto, 1996). In mammals, the VLDLR has a broad tissue distribution with a predominant MATERIALS AND METHODS expression in the central nervous system (Bujo and Yamamoto, Molecular biology 1996; May et al., 2005), suggesting that its primary function is Coding sequences for Xenopus mPRβ (NCBI Reference Sequence: separate from lipoprotein metabolism. In fact, VLDLR-knockout NM_001085861.1) and Xenopus VLDLR (GenBank: BC070552.1) were mice do not exhibit any defect in lipid homeostasis (Frykman et al., synthetized including or not including sequences for GFP (for mPR) and 1995), but rather exhibit neurodevelopmental defects reminiscent of mCherry (Ch) (for VLDLR) at the N- or C-terminus. These were then cloned in pSGEM by Mutagenex Inc. pSGEM-TMEM-mCherry has been a /Disabled-like disruption of neuronal migration – (Trommsdorff et al., 1999). Consistent with this, the VLDLR acts previously described (El-Jouni et al., 2007; Yu et al., 2009). GalNac GFP was obtained from Brian Storrie at the University of Arkansas, Fayetteville, as a canonical receptor for Reelin, and mediates its signaling ’ AR (Storrie et al., 1998), and subcloned into pSGEM. RNAs for all the functions (D Arcangelo et al., 1999; Hiesberger et al., 1999; clones were produced by in vitro transcription after linearizing the vectors Ranaivoson et al., 2016; Trommsdorff et al., 1999). Naturally with NheI using the mMessage mMachine T7 kit (Ambion). For VLDLR occurring mutations in humans strongly corroborate the role of knock down, the sense and antisense oligonucleotides sequences used were VLDLR in the brain (Ali et al., 2012; Boycott et al., 2005; Moheb as follows: sense, 5′-GATGGGAGTGTGATGGAGAC-3′; antisense, 5′- et al., 2008; Ozcelik et al., 2008; Türkmen et al., 2008). Unlike GTCTCCATCACACTCCCATC-3′. Relative expression of VLDLR and mammals, the chicken VLDLR expression is restricted to the oocyte mPR were assessed by quantitative real-time PCR (qRT-PCR). Concomitant (Bujo et al., 1994; Bujo and Yamamoto, 1996), and functions quantification of Xenopus ornithine decarboxylase mRNA transcripts Š primarily in VLDL and vitellogenin uptake to support yolk (xODC) were used to normalize mRNA transcript levels ( indelka et al., accumulation and oocyte growth (Bujo et al., 1994). Consistent 2006). The forward (F) and reverse (R) primer sequences are as follows: VLDLR: F, 5′-CGATGGGAGTGTGATGGAG-3′;R,5′-CTGCATTTG- with this, mutations in the chicken VLDLR cause female sterility CACAGTCACG; mPR: F, 5′-CCTGTTGTCCACCGGATAGT-3′;R, and severe hyperlipidemia (Bujo et al., 1995; Stifani et al., 1990). 5′-GGTGACCGTGCCCTATAAAA-3′; xODC: F, 5′-GCCATTGTGAA- In Xenopus, the VLDLR is involved in yolk accumulation in GACTCTCTCCATTC-3′,R,5′-TTCGGGTGATTCCTTGCCAC-3′. oocytes. However, based on its broad expression pattern (Okabayashi et al., 1996), it is likely to mediate other signaling Xenopus oocyte preparation and protein expression functions in the frog, similar to what is observed in mammals. Stage VI Xenopus laevis oocytes were obtained as previously described Opresko and Wiley showed a single class of low affinity (Machaca and Haun, 2002). Animals were handled according to Weill (1.3×10−6 M) and high specificity VLDLR in the Xenopus oocyte Cornell Medicine College IACUC approved procedures (protocol #2011- with an estimated internalization rate of 2×10−3 s−1 (Opresko and 0035). The oocytes were used 24 to 72 h after harvesting. Oocytes were Wiley, 1987). They further showed that the t of recovery of injected with RNAs or sense or antisense oligonucleotides and kept at 18°C 1/2 – trypsin-digested VLDLR at the PM is ∼2 h. This indicates rapid for 1 3 days after injection to allow for protein expression or efficient RNA degradation. After progesterone treatment, GVBD was recorded on a recycling of the VLDLR at the PM (within minutes), and its dissecting microscope by the appearance of a white spot at the animal pole. replenishment through a large intracellular pool of VLDLR that For the staining with WGA and P4–BSA–Fluorescein, oocytes that were supports the steady-state levels of VLDLR at the PM (Opresko and completely denuded from attached follicular cells were selected by negative Wiley, 1987). staining with Hoescht 33342 (Life Technologies) and were used for the P4– In this study, we extend the role of the VLDLR beyond BSA–Fluorescein staining experiments. lipoprotein uptake and signaling in the brain, to include for the first time a chaperone-trafficking function for mPR. We show that Oocytes crosslinking and immunoprecipitation the VLDLR is required for the transit of mPR from the ER to the Oocytes were incubated for 1 h at 4°C in freshly made ice-cold 10 mM Golgi. The intracellular distribution of the VLDLR in mammalian NHS-acetate in crosslinking buffer (10 mM HEPES pH 8.0, 150 mM NaCl, cells is reminiscent of what we observe here in the oocyte, with both 2 mM MgCl2), and then transferred to ice-cold freshly made 1 mM ER and Golgi localization (Wagner et al., 2013), suggesting a dithiobis(succinimidyl propionate) in crosslinking buffer for 30 min at 4°C. similar trafficking chaperone role in these cells. In the absence of The crosslinking reaction was stopped by incubating the oocyte for 5 min in binding solution containing 5 mM HEPES, pH 7.6, 5 mM Tris-HCl, VLDLR, mPR is enriched in the ER and is unable to traffic to the Journal of Cell Science

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100 mM NaCl, 2 mM KCl and 2 mM MgCl2. Oocytes were then lysed in IP Oocyte P4–BSA–fluorescein staining and imaging solution (30 mM HEPES, 100 mM NaCl, pH 7.5) containing protease and Oocytes were first injected with VLDLR sense or antisense nucleotides, and phosphatase inhibitors (5 µl/oocyte). Lysates were cleared of yolk by incubated for 24 or 48 h. Oocytes were fixed at 4°C for 1 h in Ringer (in centrifugation at 1000 g three times for 10 min each at 4°C. For the mM: 96 NaCl, 2.5 KCl, 1.8 CaCl2, 2 MgCl2, 10 HEPES, pH 7.4) containing proteomics studies, lysates were then immunoprecipitated with the anti-GFP 10% ethanol and BSA (8×10−7 M). Oocytes were then incubated at 4°C for microbeads (1 µl/oocyte) using the GFP isolation kit (MACS Miltenyi 2 h in Ringer with 10% ethanol containing P4–BSA–Fluorescein Biotec) as per the manufacturer’s instructions. For the co- (8×10−7 M) (Sigma-Aldrich), followed by three washes for 5 min each in immunoprecipitation studies with GFP-tagged mPR and mCherry-tagged Ringer. Oocytes were then imaged using a Zeiss LSM710 microscope with a VLDLR, lysates were first solubilized with 4% NP40 at 4°C for 2 h, 25× objective with the pinhole fully open by exciting the fluorescein followed by 15 min centrifugation at 14,000 rpm (18,400 g) at 4°C before fluorophore with the 488 nm laser and using the same master gain. ImageJ being subjected to immunoprecipitation with anti-GFP microbeads. was then used to calculate the fluorescence in a specific region of interest (ROI). ROIs of the same dimensions were used for all the oocytes. The Western blots average fluorescence of P4-BSA-FITC from uninjected oocytes was used to Cells were dounced in MPF lysis buffer [80 mM β-glycerophosphate, normalize the P4–BSA–FITC intensity in the different conditions. 20 mM Hepes (pH 7.5), 15 mM MgCl2, 20 mM EGTA, 1 mM Na- Vanadate, 50 mM NaF, 1 mM DTT, 1 mM PMSF and 0.1% protease Electrophysiology Inhibitor (Sigma)] and centrifuged twice at 1000 g for 10 min each at 4°C to The SOCE-induced Cl− currents were recorded using a standard two- remove yolk granules. Supernatants were run on 4–12% SDS-PAGE gels, electrode voltage-clamp recording technique. Recording electrodes were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore), filled with 3 M KCl and coupled to a Geneclamp 500B controlled with blocked for 1 h at room temperature with 5% milk in TBS-T buffer (150 mM pClamp 10.5 (Axon Instruments). Ca2+-activated Cl− currents were NaCl, 20 mM Tris-HCl pH 7.6 and 0.1% Tween 20) and then incubated recorded using a previously described ‘triple jump’ protocol (Courjaret overnight at 4°C in 3% BSA in TBS-T with one of the following primary and Machaca, 2016). Membrane capacitance was monitored using the built- antibodies: anti-GFP (1:1000, Living Colors, Cat# 632381), anti-mCherry in routine from pClamp to measure the cell membrane parameters. The cells and anti-RFP (1:1000, Living Colors, Cat# 632543; 1:1000, Abcam, Cat# were continuously superfused with Ringer buffer during voltage-clamp ab62341, respectively), anti-tubulin (1:10,000, Cell Signaling, Cat# 3873), experiments using a peristaltic pump. anti-phospho-MAPK (ERK1/2) (1:5000, Cell Signaling, Cat# 9106) and anti-phospho-Cdc2 (1:1000, Cell Signaling, Cat# 9111). Blots were Statistics washed three times with TBS-T and probed for 1 h with horseradish Values are given as means±s.e.m. Statistical analysis was performed when peroxidase (HRP)-conjugated secondary antibody (1:10,000; Jackson required by using a paired or unpaired Student’s t-test or ANOVA test. ImmunoResearch) (for mCherry and Cdc2), or for 1 h with IRDye® 800 P values are indicated as follows: *P<0.05, **P<0.01, ***P<0.001, and ns, and 680-conjugated secondary antibodies (1:10,000; for GFP, RFP, MAPK not significant. Each experiment is repeated at least three times from three and tubulin). The western blots were visualized by measuring either the independent donor females. chemiluminescence intensity from the peroxidase using the Immun-Star- HRP Chemiluminescent system (Bio-Rad), or by quantitative analysis using Acknowledgements the LiCor Odyssey Clx Infrared Imaging system. We thank the Proteomic Core at Weill Cornell Medicine Qatar for its support. The Core is supported by the BMRP program funded by Qatar Foundation. Proteomics Elutes obtained after GFP binding and immunoprecipitation were trypsin Competing interests digested and dimethyl labeled, with peptides from the cytosolic GFP–mPR The authors declare no competing or financial interests. immunoprecipitation (denoted ‘Cy’) labeled with the light isotope and those from the plasma membrane mPR-GFP immunoprecipitation (denoted ‘PM’) Author contributions labeled with the heavy isotope. The mentioned labeling resulted in an Conceptualization: N.N., K.M.; Methodology: N.N., M.D., R.C., R.H., J.G.; amino acid mass increase of 28 Da per primary amine on a peptide for Validation: N.N.; Formal analysis: N.N., J.G., K.M.; Investigation: N.N., M.D., R.C., the light isotope, whereas an incorporation of the heavy isotope results R.H., R.M.; Resources: K.M.; Data curation: N.N., K.M.; Writing - original draft: N.N.; in a mass increase of 36 Da. Dimethyl-labeled peptides from both Writing - review & editing: K.M.; Visualization: K.M.; Supervision: K.M.; Project administration: K.M.; Funding acquisition: K.M. immunoprecipitations were then mixed together and analyzed by mass spectrometry, allowing quantitative measurement of the differential enrichment of each detected peptide in the IPs by analyzing the ratio of Funding This work was funded by the Qatar National Research Fund (QNRF) (NPRP 7-709- heavy to the light isotope (PM:Cy). The proteomics analysis was conducted 3-195). Additional support for the authors comes from the Biomedical Research by the WCM-Q proteomic core. The experiment was repeated three times. Program (BMRP) at Weill Cornell Medical College in Qatar, a program funded by the Qatar Foundation. The statements made herein are solely the responsibility of the mPR and VLDLR imaging and quantification authors. Confocal imaging of live oocytes was performed using a LSM710 microscope (Zeiss, Germany) fitted with a Plan Apo 63×/1.4 NA oil Supplementary information immersion objective. Z-stacks were taken in 0.5 µm sections using a 1 Airy Supplementary information available online at unit pinhole aperture. Images were analyzed using ZEN 2008 (Zeiss) or http://jcs.biologists.org/lookup/doi/10.1242/jcs.212522.supplemental ImageJ software. To measure the distribution of mPR and VLDLR at the cell membrane, TMEM–mCherry or WGA were used as membrane markers. For References each oocyte, the percentage of mPR and VLDLR at the plasma membrane Ali, B. R., Silhavy, J. L., Gleeson, M. J., Gleeson, J. G. and Al-Gazali, L. (2012). A was calculated by analyzing the intensity of fluorescence distribution missense founder mutation in VLDLR is associated with Dysequilibrium through a z-stack of images, where we conservatively used two focal planes Syndrome without quadrupedal locomotion. BMC Med. Genet. 13, 80. Bandyopadhyay, A., Bandyopadhyay, J., Choi, H.-H., Choi, H.-S. and Kwon, below the peak of TMEM–mCherry fluorescence, or the low point of WGA H.-B. (1998). Plasma membrane mediated action of progesterone in fluorescence, as a reference to mark the end of the plasma membrane. The amphibian (Rana dybowskii) oocyte maturation. Gen. Comp. Endocrinol. 109, data was normalized to the average mPR membrane percentage from the 293-301. control condition. For colocalization analysis, GFP, mCherry or WGA Bayaa, M., Booth, R. A., Sheng, Y. and Liu, X. J. (2000). The classical fluorescence intensities were plotted as a function of the corresponding z- progesterone receptor mediates Xenopus oocyte maturation through a stack sections (µm). nongenomic mechanism. Proc. Natl. Acad. Sci. USA 97, 12607-12612. Journal of Cell Science

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