© 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs231886. doi:10.1242/jcs.231886

RESEARCH ARTICLE Depletion of phosphatidylinositol 4-phosphate at the Golgi translocates K-Ras to mitochondria Taylor E. Miller1, Karen M. Henkels1, Mary Huddleston2, Richard Salisbury2, Saber M. Hussain2, Atsuo T. Sasaki3 and Kwang-Jin Cho1,*

ABSTRACT CAAX endopeptidase 1 (RCE1) then removes the AAX tripeptide, Ras proteins are small GTPases localized to the plasma membrane followed by the methylation of the now C-terminal prenylated Cys (PM), which regulate cellular proliferation, apoptosis and differentiation. by isoprenylcysteine carboxyl methyltransferase (ICMT) (Clarke After a series of post-translational modifications, H-Ras and N-Ras et al., 1988; Gutierrez et al., 1989). N-Ras, H-Ras and K-Ras4A, the traffic to the PM from the Golgi via the classical exocytic pathway, alternative K-Ras splicing variant, are further modified with the but the exact mechanism of K-Ras trafficking to the PM from the ER is addition of palmitic acids on one or two other Cys residues located not fully characterized. ATP5G1 (also known as ATP5MC1) is one in the HVR (Hancock et al., 1989), allowing Ras to interact with and localize to the PM. K-Ras4B (hereafter, K-Ras) is unique in that it of the three proteins that comprise subunit c of the F0 complex of the mitochondrial ATP synthase. In this study, we show that has a single farnesyl chain preceded by a polybasic domain of six overexpression of the mitochondrial targeting sequence of ATP5G1 Lys residues (Hancock et al., 1990). The strong positive charge of perturbs glucose metabolism, inhibits oncogenic K-Ras signaling, and this polybasic domain allows K-Ras to interact with anionic redistributes phosphatidylserine (PtdSer) to mitochondria and other phospholipids in the PM through electrostatic interaction. Depletion endomembranes, resulting in K-Ras translocation to mitochondria. of phosphatidylserine (PtdSer) from the inner PM leaflet or acute 2+ Also, it depletes phosphatidylinositol 4-phosphate (PI4P) at the Golgi. neutralization of PM electrostatic potential by Ca influx results in Glucose supplementation restores PtdSer and K-Ras PM localization rapid dissociation of K-Ras from the PM (Cho et al., 2016, 2012; – and PI4P at the Golgi. We further show that inhibition of the Golgi- Yeung et al., 2008). Furthermore, K-Ras PM interaction can be localized PI4-kinases (PI4Ks) translocates K-Ras, and PtdSer to regulated through K-Ras phosphorylation, with phosphorylation at mitochondria and endomembranes, respectively. We conclude that residue Ser181 by protein kinase C or protein kinase G causing PI4P at the Golgi regulates the PM localization of PtdSer and K-Ras. mislocalization of K-Ras from the PM to endomembranes, including mitochondria (Bivona et al., 2006; Cho et al., 2016). This article has an associated First Person interview with the first author Both H-Ras and N-Ras are palmitoylated at the Golgi by a of the paper. palmitoylacyltransferase (Swarthout et al., 2005), where they are trafficked to the PM through the classical secretory pathway KEY WORDS: K-Ras, Phosphatidylserine, Phosphatidylinositol (Apolloni et al., 2000; Choy et al., 1999). H-Ras and N-Ras proteins 4-phosphate, Phosphatidylinositol 4-kinase, Golgi, Mitochondria undergo a cycle of palmitoylation and depalmitoylation, which allows them to cycle between endomembrane and PM (Lin and INTRODUCTION Conibear, 2015; Rocks et al., 2010, 2005). While the mechanism for Ras proteins are small GTPases that operate like molecular switches K-Ras trafficking from the ER to the PM is not fully elucidated, for cell proliferation, migration and apoptosis (Hancock, 2003). previous studies have implicated microtubules and possibly The three ubiquitously expressed Ras isoforms in mammalian mitochondria as having a role (Chen et al., 2000; Thissen et al., cells, K-Ras, N-Ras and H-Ras, are highly homologous in sequence 1997; Wang and Deschenes, 2006). K-Ras PM maintenance except for the C-terminal 20 amino acid residues, called the requires the chaperone protein phosphodiesterase δ (PDEδ, also hypervariable region (HVR). Post-translational modifications occur known as PDE6D). Cytosolic PDEδ binds K-Ras endocytosed from at the HVR and are important for Ras trafficking to and interacting the PM and releases K-Ras to perinuclear membranes in an Arl2- with the plasma membrane (PM), where the Ras family activates dependent manner. K-Ras then electrostatically interacts with the its downstream effectors (Hancock et al., 1989, 1990). Newly recycling endosome (RE) and returns to the PM (Chandra et al., synthesized Ras proteins are prenylated by farnesyltransferase 2012; Schmick et al., 2014). (FTase), which allows Ras to attach to the cytosolic leaflet of the ER Mitochondrial ATP synthase is a multimeric protein consisting of (Gutierrez et al., 1989; Hancock et al., 1989). RAS converting two linked complexes, F0 and F1 (Jonckheere et al., 2012). Each ATP synthase molecule is anchored to the inner mitochondrial membrane through the F0 complex, with the catalytic F1 core 1Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, OH 45435, USA. 2Human Signatures Branch, extending out into the mitochondrial matrix (Jonckheere et al., Human-Centered ISR Division, Airman Systems Directorate, 711 Human 2012). Protons from the intermembrane space funnel through the Performance Wing, Air Force Research Laboratory, Wright Patterson Air Force proton channel of the F complex, which causes the c-subunit Base, OH 45433, USA. 3Division of Hematology and Oncology, Department of 0 Internal Medicine, University of Cincinnati, Cincinnati, OH 45267, USA. oligomer ring to rotate. This rotation confers conformational changes to the structure of F1 that results in the conversion of *Author for correspondence ([email protected]) ADP+Pi to ATP (Boyer, 2000). In mammals, subunit c is encoded K.-J.C., 0000-0002-2234-9292 by three different genes (ATP5MC1, ATP5MC2, ATP5MC3) yielding three protein isoforms, which differ only in their

Received 13 March 2019; Accepted 12 July 2019 mitochondrial targeting peptide sequences (Dyer et al., 1989; Journal of Cell Science

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Dyer and Walker, 1993; Yan et al., 1994). The mitochondrial whereas the PM localization of K-Ras4AG12V (Cho et al., 2015; targeting sequence of one of these isoforms, ATP5G1 (also known Tsai et al., 2015), and the PM and Golgi localization of mGFP–H- as ATP5MC1), is used as a mitochondrial marker (Guy et al., 2002; RasG12V (Roy et al., 2005) were not perturbed (Fig. 1B). K-Ras Vives-Bauza et al., 2010). In this study, we discovered that translocates to mitochondria through PKC-mediated K-Ras overexpression of the mitochondrial targeting sequence of ATP5G1 phosphorylation at Ser181 (Bivona et al., 2006). To test whether translocates K-Ras and PtdSer to mitochondria and endomembranes, K-Ras translocation to mitochondria in cells overexpressing respectively, by mechanisms regulating phosphatidylinositol ATP5G1(1–67) is through phosphorylation of Ser181, we 4-phosphate (PI4P) contents at the Golgi. generated MDCK cells stably co-expressing ATP5G1(1–67)–RFP and a mGFP–K-RasG12V S181A mutant (Bivona et al., 2006; Cho RESULTS et al., 2016). Confocal microscopy shows that K-RasG12V S181A Overexpression of ATP5G1(1–67) translocates K-Ras to is co-localized with ATP5G1(1–67)–RFP, suggesting ATP5G1(1– mitochondria 67)-mediated K-RasG12V translocation to mitochondria is Recently, we identified several classes of compounds that independent of K-Ras phosphorylation. Taken together, Fig. 1 mislocalize K-Ras from the PM to endomembranes (Cho et al., shows that ATP5G1(1–67) overexpression translocates K-Ras from 2016; Cho et al., 2012; Salim et al., 2014a,b, 2015; Tan et al., 2018; the PM to mitochondria through its HVR in an isoform-specific van der Hoeven et al., 2013, 2017). In yeast, deletion of class C VPS manner and it is independent of K-Ras phosphorylation. genes results in mitochondrial defects and an accumulation of Ras2 on mitochondrial membranes (Wang and Deschenes, 2006). ATP5G1(1–67) overexpression perturbs cellular In mammalian cells, K-Ras translocates to mitochondria through phosphatidylserine distribution PKC-mediated phosphorylation at Ser181, inducing Bcl-XL During apoptosis, cardiolipin translocates to the outer mitochondrial (also known as BCL2L1)-dependent apoptosis (Bivona et al., membrane, which changes mitochondrial surface charge and recruits 2006). These studies suggest that mitochondria are involved in proteins with positively charged amino acid residues, including K-Ras K-Ras trafficking and signaling once K-Ras is mislocalized from (Heitetal.,2011).TotestwhetherATP5G1(1–67)-mediated K-Ras the PM. To characterize whether the identified compounds translocation to mitochondria is due to apoptosis, cell lysates of translocate K-Ras to mitochondria, MDCK cells stably expressing MDCK cells stably co-expressing ATP5G1(1–67)–RFP with mGFP– a monomeric GFP (mGFP)-tagged K-RasG12V were infected with K-RasG12V or mGFP–H-RasG12V were immunoblotted to measure lentivirus expressing a mitochondrial marker, RFP-tagged ATP5G1 cleaved caspase-3 and caspase-8 levels. Caspase cleavage is an early (amino acid residues 1–67). ATP5G1 is mitochondrial F0 complex apoptosis event, before phosphatidylserine (PtdSer) externalization subunit C1 in ATP synthase, and its mitochondrial targeting (Mariño and Kroemer, 2013). Our data show that there were no sequence (amino acid residues 1–61) followed by the first six amino detectable levels of cleaved caspase-3 or caspase-8 in these cells acids of the catalytic domain (amino acid residues 62–67) is used as (Fig. 2A). Interestingly, when cells co-expressing K-RasG12V and a mitochondrial marker (Guy et al., 2002; Higuti et al., 1993; Oca- ATP5G1(1–67) were treated with staurosporine, an apoptosis- Cossio et al., 2003; Vives-Bauza et al., 2010). inducing agent, cleaved caspase-3 and caspase-8 levels were much When ATP5G1(1–67)–RFP was overexpressed, mGFP–K- greater than in cells expressing K-RasG12V alone or H-RasG12V RasG12V unexpectedly colocalized with ATP5G1(1–67)–RFP, with ATP5G1(1–67) (Fig. 2A), suggesting that ATP5G1(1–67) suggesting that K-RasG12V is translocated from the PM to overexpression in the presence of oncogenic K-Ras, but not H-Ras, mitochondria (Fig. 1A). The same observation was made in baby enhances sensitivity to staurosporine-induced apoptosis. PKC- hamster kidney cells, suggesting it is not a cell type-specific effect induced K-Ras translocation to mitochondria has previously been (Fig. S1). In control MDCK cells co-expressing mGFP–K- shown to stimulate apoptosis in a Bcl-XL-dependent manner RasG12V and mCherry–CAAX, an endomembrane marker (Cho (Bivona et al., 2006). Combining this with our data, it suggests that et al., 2012; Choy et al., 1999), K-RasG12V is localized to the PM mitochondrial K-Ras in cells overexpressing ATP5G1(1–67) confers (Fig. 1A). Furthermore, incubating MDCK cells stably expressing high sensitivity to staurosporine-induced apoptosisthrough interaction mGFP–K-RasG12V with MitoTracker or baculovirus expressing with Bcl-XL. To further test cellular apoptosis, we performed an the RFP-fused leader sequence of mitochondrial protein pyruvate annexin V binding assay. Annexin V is a non-permeable PtdSer- dehydrogenase E1 α1 subunit (PDHA1) (Sutendra et al., 2014) did binding protein that only binds to cells when PtdSer is exposed in the not disrupt K-RasG12V PM localization (Fig. 1A). To quantitate the outer PM leaflet during early apoptosis (Balasubramanian et al., 2007; extent of colocalization of mGFP–K-RasG12V with mitochondrial Segawa et al., 2014). MDCK cells stably expressing RFP alone or markers, we used Manders’ coefficient, which provides an estimate ATP5G1(1–67)–RFP were incubated with annexin V, and annexin V- of the fraction of mitochondrial markers colocalized with mGFP–K- positive cells were counted using a cytometer. Our data show that RasG12V. Manders’ coefficient for ATP5G1(1–67) showed a ATP5G1(1–67) overexpression did not have any effect on annexin V higher value than other mitochondrial markers (Fig. 1A, bottom binding (Fig. 2B), suggesting it does not induce apoptosis. Taken right of merge images). Taken together with the confocal imaging, together, our data suggest that ATP5G1(1–67) overexpression does our data suggest that overexpression of ATP5G1(1–67) translocates not induce apoptosis, and that ATP5G1(1–67)-mediated K-Ras K-RasG12V to mitochondria. mislocalization to mitochondria is independent of apoptosis. To further characterize the mechanism of ATP5G1(1–67)- PtdSer is a phospholipid concentrated in the inner PM leaflet, induced K-Ras mislocalization, we examined the localization of where its anionic head group interacts with the polybasic domain other Ras isoforms and the C-terminal hypervariable region of K- and farnesyl anchor of K-Ras, allowing K-Ras PM binding (Yeung Ras (CTK). ATP5G1(1–67)–RFP was overexpressed in MDCK et al., 2008; Zhou et al., 2017). To further validate the effect of cells stably expressing mGFP–CTK, mGFP–K-Ras4AG12V, the ATP5G1(1–67) overexpression on cellular PtdSer distribution, alternate K-Ras splicing variant (McGrath et al., 1983), or mGFP– ATP5G1(1–67)–RFP was overexpressed in MDCK cells stably H-RasG12V, and images were taken using a confocal microscope. expressing well-characterized mGFP-tagged PtdSer markers, the C2

Our data show that CTK was colocalized with ATP5G1(1–67), domain of lactadherin (LactC2) (Cho et al., 2012; Yeung et al., 2008) Journal of Cell Science

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Fig. 1. Overexpression of ATP5G1(1–67) translocates K-Ras to mitochondria. (A) To evaluate expression of mitochondrial markers, MDCK cells stably expressing mGFP–K- RasG12V were infected with lentivirus expressing ATP5G1(1–67)–RFP, incubated with modified baculovirus encoding RFP- tagged PDHA1 for 16 h, or stained with 100 nM MitoTracker Deep Red for 1 h. Cells were fixed with 4% PFA and imaged using a confocal microscope. As a control, MDCK cells stably co-expressing mGFP–K- RasG12V with endomembrane marker mCherry–CAAX were used. Values bottom right of merge panels represent mean±s.e.m. of the fraction of mitochondrial markers colocalized with mGFP–K-RasG12V calculated by Manders’ coefficient from three independent experiments. Scale bars: 10 µm. (B) MDCK cells stably co-expressing ATP5G1(1–67)–RFP with mGFP-tagged CTK, K-Ras4AG12V, H-RasG12V or K- RasG12V S181A were fixed with 4% PFA and imaged using a confocal microscope. Inserted values represent mean±s.e.m. of the fraction of ATP5G1(1–67)–RFP colocalized with mGFP-tagged Ras isoforms calculated by Manders’ coefficient from three independent experiments. Scale bars: 10 µm. All cells were maintained in complete growth medium (DMEM+10% FBS+2 mM L-glutamine).

or the PH domain of evectin-2 (also known as PLEKHB2) (evectin- Akt (pAkt) proteins. Our data show that ATP5G1(1–67)–RFP 2-PH) (Uchida et al., 2011). While LactC2 and evectin-2-PH were overexpression significantly reduced ppERK and pAkt levels in K- predominantly localized to the PM when mCherry-CAAX was co- RasG12V, and to a lesser extent in H-RasG12V cell lines (Fig. 3A). expressed, they were redistributed to mitochondria (indicated by Taken together with confocal microscopy results (Fig. 1), our data closed arrows) and other endomembranes (indicated by open arrows) demonstrate that ATP5G1(1–67) overexpression inhibits signaling of in cells overexpressing ATP5G1(1–67) (Fig. 2C,D). Taken together, oncogenic K-Ras, but not H-Ras, through PM mislocalization. our data suggest that ATP5G1(1–67) overexpression redistributes PtdSer to mitochondria and other endomembranes. Glucose supplementation returns K-Ras to the PM Efficient silencing of ATP5G1 induces mitochondrial dysfunction ATP5G1(1–67) overexpression inhibits oncogenic K-Ras such as reduction of ATP production, the number of fully assembled signal output ATP synthase molecules, mitochondrial fitness, and altered Ras proteins must interact primarily with the PM to stimulate mitochondrial morphology (Vives-Bauza et al., 2010). We therefore Raf/MEK/ERK and PI3K/Akt signaling pathways (Hancock, hypothesized that cellular ATP level is reduced in cells 2003; Willumsen et al., 1984). Thus, we studied the effect of overexpressing ATP5G1(1–67)bydisruptingmitochondrialATP ATP5G1(1–67) overexpression in oncogenic Ras signal output. Cell synthase, which results in disruption of PtdSer and K-Ras4B PM lysates from MDCK cells stably co-expressing mGFP–K-RasG12V localization. To test this, fully confluent MDCK cells grown in or mGFP–H-RasG12V together with RFP only or ATP5G1(1–67)– complete growth medium and expressing mGFP–K-RasG12V in the

RFP were immunoblotted for phosphorylated ERK (ppERK) and presence or absence of ATP5G1(1–67)–RFP were transferred to fresh Journal of Cell Science

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Fig. 2. ATP5G1(1–67) overexpression redistributes PtdSer from the PM independent of apoptosis. (A) MDCK cells stably expressing mGFP–K-RasG12V or mGFP–H-RasG12V with or without ATP5G1(1–67)–RFP were treated with 1 µM staurosporine (STS) to induce apoptosis or vehicle (DMSO) for 6 h. Cell lysates were blotted with anti-cleaved caspase-3 or caspase-8 antibodies as markers of early apoptosis. A GAPDH blot was used for a loading control. Representative blots are shown from three independent experiments. (B) MDCK cells stably expressing RFP alone (open bars) or ATP5G1(1–67)–RFP (closed bars) were incubated with FITC-conjugated annexin V, and annexin V-positive cells were counted in a cytometer. Cells treated with 1 µM STS for 6 h were used as positive controls to induce apoptosis. A difference between annexin V-treated cells with or without ATP5G1(1–67) were assessed using a Student’s t-test (N.S., not significant). (C,D) MDCK cells co-expressing mGFP–LactC2 (C) or mGFP–evectin-2-PH (D) with mCherry– CAAX or ATP5G1(1–67)–RFP were fixed with 4% PFA and imaged using a confocal microscope. A selected region indicated by the white square is shown at a higher magnification. mGFP– LactC2 or mGFP–evectin-2-PH colocalized and not colocalized with ATP5G1(1–67)–RFP are indicated by closed and open arrows, respectively. Scale bars: 10 µm. All cells were maintained in complete growth medium (DMEM+10% FBS+2 mM L-glutamine).

complete growth medium, and cell images were taken at different time were incubated with fresh complete growth medium without glucose points. Manders’ coefficients for ATP5G1(1–67) colocalization with only [(−)glucose], without pyruvate only [(−)pyruvate], or without K-RasG12V were calculated to quantitate K-RasG12V localization to glucose and pyruvate [(−)glucose/pyruvate] for 2 h. Cell images were mitochondria (Fig. 3B, values bottom right of image panels). Our data taken and Manders’ coefficients for ATP5G1(1–67) colocalization show that in cells overexpressing ATP5G1(1–67), K-RasG12V with K-RasG12 V were calculated to quantitate K-Ras localization to returned to and remained at the PM from 0.5 to 6 h after the mitochondria. Our data show that in cells incubated with (−)pyruvate supplementation, with cells at the 2 h time point showing the most K- medium, K-RasG12V returned to the PM, whereas in cells incubated Ras PM localization. However, K-RasG12V was again mislocalized with (−)glucose medium, it did not (Fig. 3C). The same results to mitochondria 24 h after the supplementation (Fig. 3B). K- were observed for PtdSer localization (Fig. 3C). MDCK cells RasG12V remained at the PM during these time points in the co-expressing mGFP–LactC2 and ATP5G1(1–67)–RFP were absence of ATP5G1(1–67)–RFP (Fig. 3B). These data suggest that in incubated with the same panel of growth media, and cell images cells overexpressing ATP5G1(1–67), nutrients in complete growth were taken. Our data show that in cells incubated with (−)pyruvate medium can rapidly correct K-Ras PM localization, but once these and complete growth medium, but not in (−)glucose medium, nutrients are depleted, K-Ras translocates back to mitochondria. To LactC2 returned to the PM 2 h after the incubation (Fig. 3C). Taken further elucidate the nutrients required for K-Ras PM localization in together, these data suggest that sufficient cellular glucose levels are cells overexpressing ATP5G1(1–67), fully confluent MDCK cells required for K-Ras and PtdSer PM localization in cells stably co-expressing mGFP–K-RasG12V and ATP5G1(1–67)–RFP overexpressing ATP5G1(1–67). Journal of Cell Science

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Fig. 3. Glucose is required for the PM localization of K-RasG12V and LactC2 in cells overexpressing ATP5G1(1–67). (A) Cell lysates of MDCK cells stably co-expressing mGFP–K-RasG12V or mGFP–H-RasG12V with RFP alone (−) or ATP5G1(1–67)–RFP (+) were immunoblotted for phospho-ERK, phospho-Akt (S473), and mGFP–RasG12V, and quantified (mean±s.e.m.) from three independent experiments. Significant differences between cells not expressing (−) or expressing (+) ATP5G1(1–67) were assessed using Student’s t-test (*P<0.05; ***P<0.001). Representative blots of are shown, with total ERK, total Akt and actin used as loading controls. (B) MDCK cells stably expressing mGFP–K-RasG12V in the presence (+) or absence (−) of ATP5G1(1–67)–RFP were maintained in complete growth medium (DMEM+10% FBS+2 mM L-glutamine). When cells reached full confluence, the growth medium was replaced with fresh complete growth medium and cells were further incubated for indicated time points. Cells were fixed with 4% PFA and imaged using a confocal microscope. Inserted values represent mean±s.e.m. of the fraction of ATP5G1(1–67)–RFP colocalized with mGFP–K-RasG12V calculated by Manders’ coefficient from three independent experiments. Shown are representative mGFP–K-RasG12V images. Scale bars: 10 µm. (C) MDCK cells stably co-expressing ATP5G1(1–67)–RFP with mGFP–K-RasG12V, or mGFP–LactC2 were maintained in complete growth medium (DMEM+10% FBS+2 mM L-glutamine). When cells reached full confluence, the growth medium was left unchanged (no supplement) or replaced with fresh complete growth medium without pyruvate only [(−)pyruvate], without glucose only [(−)glucose], without glucose and pyruvate [(−)glucose/pyruvate], or in complete growth medium [(+)glucose/pyruvate], and incubated for another 2 h. Cells were fixed with 4% PFA and imaged using a confocal microscope. Inserted values represent mean±s.e.m. of the fraction of ATP5G1(1–67)–RFP colocalized with mGFP–K-RasG12V or mGFP–LactC2 calculated by Manders’ coefficient from three independent experiments. Shown are representative mGFP–K-RasG12V and mGFP–LactC2 images. Scale bars: 10 µm.

ATP5G1(1–67) overexpression increases glucose RFP were cultured in complete growth medium for 48 h, then consumption and inhibits glycolysis incubated with fresh complete growth medium for another 2 h. The To characterize the effect of ATP5G1(1–67) on glucose growth medium and cell lysates were harvested before (basal) and metabolism, we measured glucose consumption, cellular ATP after (refeed) the medium was changed to measure glucose levels, levels, and the ADP/ATP ratio. MDCK cells stably co-expressing and cellular ATP levels and the ADP/ATP ratio, respectively. We mGFP–K-RasG12V together with RFP only or ATP5G1(1–67)– found that the glucose level was significantly reduced in cells Journal of Cell Science

5 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs231886. doi:10.1242/jcs.231886 overexpressing ATP5G1(1–67) in the basal condition, suggesting level (Fig. 4A). For cellular ATP levels, no difference was observed that cells overexpressing ATP5G1(1–67) consumed more glucose between the cell lines in the basal condition, whereas the change of (Fig. 4A). After the medium was changed, glucose in the growth medium slightly reduced ATP levels in both cell lines (Fig. 4B). We medium was significantly increased in both cell lines to a similar further found that the ADP/ATP ratio was significantly lower in

Fig. 4. ATP5G1(1–67) overexpression increases glucose consumption and inhibits glycolysis.(A–C) MDCK cells stably co-expressing K-RasG12V with RFP alone (−) or ATP5G1(1–67)–RFP (+) were grown to full confluence in complete growth medium. The growth medium was left unchanged (basal) or replaced with fresh complete growth medium (refeed), and cells were incubated for another 2 h. The growth medium were collected for glucose consumption assay (A), and cell lysates were harvested for cellular ATP assay (B) and calculation of the ADP/ATP ratio (C). (D–N) MDCK cells stably co-expressing K- RasG12V with RFP alone (−) or ATP5G1(1–67)–RFP (+) were grown to full confluence in complete growth medium. Medium was replaced with fresh XF assay medium, which does not contain glucose and pyruvate, and cells further incubated at 37°C in 0% CO2 for 45–60 min. Using a Seahorse Analyzer, oxygen consumption rate (OCR) was measured to analyze mitochondrial respiration after treating cells with compounds at different time points (A, oligomycin; B, FCCP; C, rotenone and antimycin A) (D–I), or extracellular acidification rate (ECAR) was measured to analyze glycolytic rate after treating cells with compounds at different time points (A, glucose; B, oligomycin; C, 2-deoxy-D-glucose) (J–N). Significant differences between cells not expressing (−) or expressing (+)

ATP5G1(1–67) were assessed using Student’s t-test (N.S., not significant; *P<0.05; **P<0.01; ***P<0.001). Journal of Cell Science

6 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs231886. doi:10.1242/jcs.231886 cells overexpressing ATP5G1(1–67) in the basal condition cells overexpressing ATP5G1(1–67) is induced by PtdSer content (Fig. 4C). Since cellular ATP level was unchanged in both cell depletion at the PM. lines (Fig. 4B), our data suggest a reduction of ADP level in cells To further examine whether K-Ras translocation to mitochondria is overexpressing ATP5G1(1–67) in the basal condition. After mediated by PtdSer accumulation at mitochondria, we depleted medium was changed, the ADP/ATP ratio was reduced in both PtdSer content at mitochondria by overexpressing phosphatidylserine cell lines to a similar level (Fig. 4C). decarboxylase (PISD), an enzyme that converts PtdSer to To further characterize the effect of ATP5G1(1–67) overexpression phosphatidylethanolamine in the inner mitochondrial membrane on glucose consumption and mitochondrial ATP synthesis, we (Percy et al., 1983). Briefly, MDCK cells stably co-expressing measured the glycolytic rate and mitochondrial respiration using ATP5G1(1–67)–RFP and mGFP–K-RasG12V or mGFP–LactC2 a Seahorse Analyzer. Briefly, MDCK cells stably co-expressing were infected with lentivirus expressing PISD or an empty vector, and mGFP–K-RasG12V together with RFP only or ATP5G1(1–67)–RFP cell images were taken using a confocal microscope. Manders’ were grown to full confluence in complete growth medium. Cells were coefficients for colocalization of K-RasG12V or LactC2 with then incubated with XF assay medium, which does not contain ATP5G1(1–67) were calculated to quantitate the fraction of glucose, in a non-CO2 37°C incubator for 45–60 min. Oxygen K-RasG12V or LactC2 localized to the mitochondria. Our data consumption rate (OCR) and extracellular acidification rate (ECAR), show that LactC2 was distributed to endomembranes including indicators of the mitochondrial respiration and glycolytic rate, mitochondria in control cells and cells overexpressing PISD respectively, were measured after adding compounds that regulate (Fig. 5B), suggesting that PtdSer distribution was not affected on mitochondrial activity or glycolysis at indicated time points. We PISD overexpression. However, Manders’ coefficient values were found that OCRs in all parameters of mitochondrial respiration that we significantly reduced from 0.31 to 0.22 on PISD overexpression, measured were unchanged in the presence or absence of ATP5G1(1– indicating that the fraction of LactC2 localized to mitochondria was 67) after adding compounds regulating mitochondrial activity decreased from 31% to 22% when PISD was overexpressed (Fig. 5C). (Fig. 4D–I). These data suggest that the mitochondrial respiration, These data suggest that PISD overexpression partially reduces PtdSer including ATP production, is unaffected by ATP5G1(1–67) content at mitochondria. PISD overexpression also redistributed K- overexpression, consistent with the cellular ATP levels (Fig. 4B). RasG12V to endomembranes, including mitochondria, while it was However, we observed that ECAR was significantly inhibited in predominantly localized to mitochondria in control cells (Fig. 5D). cells overexpressing ATP5G1(1–67) after adding oligomycin Manders’ coefficients were also significantly reduced from 0.33 to (Fig. 4J), which shifts the energy production from mitochondria 0.25 (Fig. 5E), indicating that the fraction of K-RasG12V localized to glycolysis. Further analyses show that glycolysis, glycolytic to mitochondria was decreased from 33% to 25% when PtdSer capacity and glycolytic reserve were also significantly inhibited in content at mitochondria was reduced. PISD overexpression did not cells overexpressing ATP5G1(1–67) (Fig. 4K–N). Glycolytic disrupt the PM localization of K-RasG12V and LactC2 in cells not capacity measures the maximum rate of glycolysis, whereas expressing ATP5G1(1–67) (Fig. S2C). Taken together with glycolytic reserve indicates the capability of glycolytic response to phospholipid supplementation experiments, our data suggest that K- an increased ATP demand (Mookerjee et al., 2016). Taken Ras translocation to mitochondria is induced by PtdSer accumulation together, our data indicate that ATP5G1(1–67) overexpression at mitochondria in cells overexpressing ATP5G1(1–67). enhances cellular glucose consumption while inhibiting the glycolytic process. Phosphatidylinositol 4-phosphate distribution is perturbed in cells overexpressing ATP5G1(1–67) K-Ras translocation to mitochondria is mediated by PtdSer To characterize the effects of ATP5G1(1–67) overexpression on accumulated at mitochondria other phospholipids, we examined cellular localization of We showed that ATP5G1(1–67) overexpression redistributes PtdSer phospholipid markers in the presence or absence of ATP5G1(1– from the PM (Fig. 2C,D). To test whether K-Ras mislocalization is 67). ATP5G1(1–67)–RFP was overexpressed in MDCK cells stably induced by PtdSer redistribution, we performed lipid addback expressing the following mGFP-tagged phospholipid markers and experiments. MDCK cells stably co-expressing ATP5G1(1–67)– confocal microscopy was performed; the PH domain of FAPP1 (also RFP with mGFP–LactC2 or mGFP–K-RasG12V were incubated known as PLEKHA3) (FAPP1-PH) or the P4M domain of SidM with 10 µM PtdSer or phosphatidylcholine (PtdCho), and cells were (P4M-SidM) for phosphatidylinositol (PI) 4-phosphate (PI4P) imaged at different time points. Manders’ coefficients for (Hammond et al., 2014), PH-Akt for PI(3,4)P2 or PI(3,4,5)P3 ATP5G1(1–67) colocalization with K-RasG12V or LactC2 were (Franke et al., 1997; James et al., 1996), tandem FYVE domains of calculated to quantitate the mitochondria localization. Our data EEA1 (2×FYVE) for PI3P (Stenmark et al., 1996), PH-PLCδ1 for show that LactC2 predominantly decorated the PM after 15 min PI(4,5)P2 (Garcia et al., 1995), and the phosphatidic acid-binding of PtdSer supplementation, which suggests that the exogenous domain of Spo20 (PASS) for phosphatidic acid (Zhang et al., 2014). PtdSer was displayed on the inner leaflet of the PM (Fig. 5A), In the absence of ATP5G1(1–67), PI4P probes were localized to the consistent with the time frame reported previously (Cho et al., 2012, PM and Golgi, consistent with a previous study (Hammond et al., 2015). An hour after PtdSer supplementation, however, LactC2 2014), whereas Golgi, but not PM (indicated by arrows) localization was redistributed to endomembranes, including mitochondria, as was perturbed in cells overexpressing ATP5G1(1–67) (Fig. 6A). The indicated by increased Manders’ coefficients. A similar observation Golgi localization of the PI4P probes was restored after cells were was made with K-RasG12V. PtdSer supplementation restored incubated with fresh complete growth medium for 2 h (Fig. 6A). To K-RasG12V PM localization within 15 min (Fig. 5A), but further characterize the effect of glucose depletion on PI4P K-RasG12V was translocated back to mitochondria 60 min after localization, MDCK cells stably expressing mGFP–P4M-SidM the supplementation. Supplementation with PtdCho, which is not only were incubated in growth medium with or without glucose for required for LactC2 and K-Ras PM binding (Cho et al., 2015), did 6 h. Our data show that glucose depletion perturbed PI4P content at not correct LactC2 and K-RasG12V mislocalization (Fig. S2A,B). the Golgi without disrupting PI4P at the PM (Fig. 6B). Cellular

Taken together, our data suggest that K-Ras PM mislocalization in localization of other phospholipid markers was unaffected in cells Journal of Cell Science

7 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs231886. doi:10.1242/jcs.231886

Fig. 5. K-Ras translocation to mitochondria is induced by mitochondrial PtdSer. (A) MDCK cells stably co-expressing ATP5G1(1–67)– RFP with mGFP–LactC2 or mGFP–K- RasG12V were supplemented with 10 µM exogenous PtdSer or PtdCho (Fig. S2) in complete growth medium without glucose and further incubated for indicated time points. Cells were fixed with 4% PFA and imaged using a confocal microscope. Inserted values represent mean±s.e.m. of the fraction of ATP5G1(1–67)–RFP colocalized with mGFP–LactC2 or mGFP–K-RasG12V calculated by Manders’ coefficient from three independent experiments. Shown are representative images of mGFP–LactC2 and mGFP–K-RasG12V. Scale bars: 10 µm. (B–E) MDCK cells stably co- expressing ATP5G1(1–67)–RFP with mGFP–LactC2 (B) or mGFP–K- RasG12V (D) were infected with lentivirus expressing PISD or an empty vector. Cells were maintained in complete growth medium. Cells were fixed with 4% PFA and imaged using a confocal microscope. A selected region indicated by the white square is shown at a higher magnification. K-RasG12V and LactC2 colocalized and not colocalized with ATP5G1(1–67) are indicated by closed and open arrows, respectively. Inserted values and the graphs represent mean±s.e.m. of the fraction of mGFP–LactC2 (C) or mGFP– K-RasG12V (E) colocalized with ATP5G1(1–67)–RFP calculated by Manders’ coefficient in cells overexpressing an empty vector or PISD from three independent experiments. The difference between control (empty) and cells overexpressing PISD (PISD) cells was assessed using Student’s t-test (**P<0.01; ****P<0.0001). Scale bars: 10 µm.

overexpressing ATP5G1(1–67) (Fig. 6C). Taken together with the at the Golgi redistributes PtdSer to mitochondria and other glucose consumption data (Fig. 4), these data suggest that endomembranes, which translocates K-Ras to mitochondria. To ATP5G1(1–67) overexpression depletes PI4P at the Golgi by test this, we examined K-RasG12V and LactC2 localization after depleting cellular glucose. inhibition of Golgi-localized PI4Ks. There are four PI4K isoforms: PI4K2A, PI4K2B, PI4KA and PI4KB. Of those, PI4K2A and PI4KB Inhibition of the Golgi-localized PI4 kinases mislocalizes are localized predominantly at the Golgi complex (Wang et al., 2003; K-Ras and PtdSer from the PM Weixel et al., 2005). Synthetic inhibitor PIK-93 specifically inhibits PI4P is synthesized from PI or PI(4,5)P2 by PI4 kinases (PI4Ks) or PI4KB (Knight et al., 2006; Tóth et al., 2006), whereas phenylarsine inositol 5-phosphatases, respectively (Hammond et al., 2012; oxide blocks PI4K2A/B activities (Sorensen et al., 1998; Wiedemann Varnai et al., 2006). Recent studies reported that PM PI4P et al., 1996). MDCK cells stably expressing mGFP–K-RasG12V or depletion through inhibition of PM-localized PI4KA mislocalizes mGFP–LactC2 were incubated with baculovirus expressing RFP– PtdSer from the PM and translocates K-Ras to the Golgi (Chung PDHA1 and the PI4K inhibitors for 48 h. Cell images were taken et al., 2015; Gulyás et al., 2017; Moser von Filseck et al., 2015). using a confocal microscope and Manders’ coefficients for RFP– We show that ATP5G1(1–67) overexpression depletes PI4P at PDHA1 colocalization with K-RasG12V or LacC2 were calculated the Golgi without altering the PM PI4P and PI(4,5)P2 distribution, to quantitate their mitochondrial localization. Our data show and mislocalizes PtdSer and K-Ras from the PM (Figs 1, 2 and 6). that while K-RasG12V did not colocalize with RFP–PDHA1 in

Taken together, these results led usto hypothesize that PI4P depletion vehicle-treated control cells, it colocalized with RFP–PDHA1 after Journal of Cell Science

8 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs231886. doi:10.1242/jcs.231886

Fig. 6. ATP5G1(1–67) overexpression reduces PI4P at the Golgi. (A) MDCK cells stably co-expressing ATP5G1(1–67)–RFP with mGFP–FAPP1-PH or mGFP–4M-SidM were maintained in complete growth medium (DMEM+10% FBS+2 mM L-glutamine). When cells reached full confluence, the growth medium was left unchanged or replaced with fresh complete growth medium and cells were incubated for 2 h (+supplement). Cells were fixed with 4% PFA and imaged using a confocal microscope. As a control, MDCK cells stably expressing mGFP–FAPP1-PH or mGFP–P4M- SidM only [(−) ATP5G1(1–67)] maintained in complete growth medium were imaged. Shown are representative mGFP–FAPP1-PH and mGFP–P4M-SidM images. Arrows indicate the PM localization of FAPP1-PH and P4M-SidM. Scale bars: 10 µm. (B) Fully confluent MDCK cells stably expressing mGFP–P4M-SidM only were incubated in growth medium with (+glucose) or without glucose (−glucose) for 6 h. Cells were fixed with 4% PFA followed by confocal microscopy. Scale bars: 10 µm. (C) MDCK cells stably expressing phospholipid markers PH-Akt–mGFP, mGFP–2×FYVE, mGFP–PH-PLCδ1, or mGFP–PASS with or without ATP5G1(1–67)–RFP were grown to full confluence in complete growth medium, fixed with 4% PFA and imaged using a confocal microscope. Shown are representative images of the phospholipid markers. Scale bars: 10 µm.

treatment with the PI4K inhibitors at a concentration that depleted depletion reduces PI4P contents at the Golgi, but not the PM, and PI4P at the Golgi, but not at the PM (Fig. 7A; Fig. S3). In cells that PI4P depletion at the Golgi by inhibition or knockout of Golgi- expressing mGFP–LactC2, the PI4K inhibitors redistributed localized PI4Ks also translocates K-Ras and PtdSer to mitochondria LactC2 to mitochondria (indicated by closed arrows) and other and endomembranes, respectively. Taken together, our study endomembranes (indicated by open arrows) (Fig. 7B). The same demonstrates that PI4P content at the Golgi is sensitive to cellular observation was made when cells treated with the PI4K inhibitors glucose levels, and that PI4P content at the Golgi regulates the PM were stained with MitoTracker (Fig. S4). These data suggest that localization of PtdSer and K-Ras. PI4KB, one of the Golgi-localized inhibition of the Golgi-localized PI4Ks mislocalizes K-Ras and PI4Ks, and Pik1p, the yeast homolog of PI4KB, have been reported PtdSer from the PM to mitochondria and endomembranes, to localize to the Golgi and the nucleus (de Graaf et al., 2002; Strahl respectively. To further validate the role of Golgi-localized PI4Ks, et al., 2005). Under glucose depletion, Pik1p dissociates from the we knocked out PI4KB in MDCK cells stably expressing mGFP–K- Golgi and accumulates in the nucleus by interacting with 14-3-3 RasG12V or mGFP–LactC2 using CRISPR-Cas9 technology, and proteins, with this response reversed upon glucose supplementation studied the cellular localization of K-RasG12V and LactC2. (Demmel et al., 2008). Based on these studies and our own Confocal microscopy data show that when PI4KB expression was observations, we propose that glucose depletion translocates PI4KB completely abolished (Fig. 8A,B; Fig. S5), K-RasG12V and LactC2 from the Golgi to the nucleus, which, in turn, depletes PI4P content translocated to mitochondria and endomembranes, respectively at the Golgi, leading to the PM mislocalization of PtdSer and K-Ras. (Fig. 8C,D). Taken together with the PI4K inhibitor data, our data Upon glucose supplementation, PI4KB returns to and produces suggest that depletion of Golgi-localized PI4P by inhibition or PI4P at the Golgi, resulting in the return of PtdSer and K-Ras to knockout of the Golgi-localized PI4Ks translocates K-Ras and the PM. PtdSer to mitochondria and endomembranes, respectively. It is unclear how ATP5G1(1–67) overexpression promotes cellular glucose consumption while inhibiting the glycolytic DISCUSSION process. The pentose phosphate pathway (PPP) uses glucose-6- In this study, we show that overexpression of the mitochondrial phosphate (G-6-P) converted from glucose to ultimately produce targeting sequence and the leading six amino acid residues of the ribose-5-phosphate for nucleic acid synthesis, and NADPH, catalytic domain (amino acids 1–67) of ATP5G1, a subunit of important in anti-oxidant defense against reactive oxygen species mitochondrial ATP synthase, elevates cellular glucose consumption (ROS) (Patra and Hay, 2014; Stanton, 2012). It is plausible that and blocks glycolysis without disrupting mitochondrial respiration. overexpression of a truncated mutant subunit in the mitochondrial ATP5G1(1–67) overexpression also depletes PI4P at the Golgi, and ATP synthase complex may elevate ROS by stressing the redistributes PtdSer to mitochondria and other endomembranes, mitochondrial reparatory chain. This, in turn, promotes cells to resulting in K-Ras translocation to mitochondria. These effects are utilize glucose preferentially for the PPP over glycolysis to generate reversed by glucose supplementation. We further show that glucose sufficient NADPH to defend against the elevated ROS. Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs231886. doi:10.1242/jcs.231886

Fig. 7. Golgi-localized PI4K inhibition mislocalizes K-RasG12V and PtdSer to mitochondria and endomembrane, respectively. (A,B) MDCK cells stably expressing mGFP–K-RasG12V (A) or mGFP– LactC2 (B) were treated with vehicle (DMSO), 1 µM PIK-93 or 50 nM phenylarsine oxide for 48 h in the presence of modified baculovirus encoding RFP– PDHA1. Cells were fixed with 4% PFA and imaged using a confocal microscope. Inserted values are mean±s.e.m. of the fraction of RFP–PDHA1 colocalized with mGFP–K-RasG12V or mGFP– LactC2, calculated by Manders’ coefficient from three independent experiments. Selected regions indicated by the white squares are shown at a higher magnification. mGFP–K-RasG12V and mGFP– LactC2 colocalized and not colocalized with RFP– PDHA1 are indicated by closed and open arrows, respectively. Scale bars: 10 µm.

PI4P metabolism is important for PtdSer localization at the inner depleted by inhibition of Golgi-localized PI4Ks, OSBP is no longer PM leaflet. Studies in mammalian cells and yeast identified that recruited to ER–Golgi membrane contact sites, resulting in reduced oxysterol-binding protein-related proteins 5 and 8 (ORP5, ORP8, PI4P content in the ER. This, in turn, perturbs the PI4P gradient also known as OSBPL5 and OSBPL8) exchange newly synthesized necessary for the ORP5/8 machinery, resulting in PtdSer PtdSer from the ER for PI4P from the PM (Chung et al., 2015; redistribution. Moser von Filseck et al., 2015). This exchange is driven by the Here, we show that K-Ras translocation to mitochondria is synthesis of PI4P at the PM by PI4KA and the concomitant induced by PtdSer accumulated at mitochondria in cells hydrolysis of PI4P by Sac1 phosphatase in the ER to maintain a overexpressing ATP5G1(1–67) (Fig. 5). It is not fully understood PI4P gradient across the PM and ER. PI(4,5)P2 at the PM further why K-Ras is accumulated predominantly at mitochondria, while promotes the recruitment of ORP8 to the PM for transporting PI4P PtdSer is redistributed to mitochondria and other endomembranes. to the ER. Eliminating any component of this mechanism depletes We speculate that the outer mitochondrial membranes with PtdSer content in the inner PM leaflet (Chung et al., 2015; Moser increased PtdSer content upon inhibition of Golgi-localized von Filseck et al., 2015; Sohn et al., 2018). PI4P at the Golgi is PI4Ks provide a lipid environment necessary for recruitment of also transported to the ER by oxysterol binding protein (OSBP) at and interaction with the polybasic domain and farnesyl-anchor ER–Golgi membrane contact sites. OSBP exchanges Golgi PI4P for of K-Ras. When PtdSer at mitochondria is depleted by PISD sterol from the ER, and OSBP dissociates from the Golgi when overexpression, the outer mitochondrial membranes no longer Golgi PI4P levels are low (Mesmin et al., 2013). Taking these provide the lipid environment, resulting in K-Ras redistribution to studies together with our data, we propose that when Golgi PI4P is other endomembranes. Intriguingly, while depletion of PM PI4P Journal of Cell Science

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Fig. 8. Knockout of PI4KB translocates K-Ras and PtdSer to mitochondria and endomembranes, respectively. Cell lysates from MDCK cells with PI4KB knocked out by means of CRISPR-Cas9 (sgRNA2, sgRNA6), and stably expressing mGFP–K-RasG12V (A) or mGFP–LactC2 (B) were immunoblotted with anti-PI4KB antibody, with lysates from MDCK cells transfected with an empty vector as control. Cell lysates from HEK293T cells overexpressing human PI4KB (PI4KB O.E.) were used as a positive control for the antibody. A representative blot is shown from three independent experiments, and an actin blot is shown as a loading control. (C,D) PI4K knockout MDCK cells stably expressing mGFP–K- RasG12V (C) or mGFP–LactC2 (D) were incubated with baculovirus encoding RFP–PDHA1 for 48 h. Cells were fixed with 4% PFA and imaged using a confocal microscope. Inserted values are mean±s.e.m. of the fraction of RFP–PDHA1 colocalized with mGFP–K-RasG12V or mGFP–LactC2 calculated by Manders’ coefficient from three independent experiments. Selected regions indicated by the white squares are shown at a higher magnification. mGFP–K-Ras and mGFP– LactC2 colocalized and not colocalized with RFP–PDHA1 are indicated by closed and open arrows, respectively. Scale bars: 10 µm.

redistributes PtdSer from the PM, it translocates K-Ras 1-Ig; 1:5000) and GFP (66002-1-lg; 1:4000) were purchased from predominantly to the Golgi, which is diminished by the Proteintech. Anti-cleaved caspase 3 (9661; 1:1000), anti-cleaved caspase concomitant depletion of Golgi-localized PI4P (Gulyás et al., 8 (9496; 1:1000), anti-total Akt (2920; 1:1000), anti-total ERK (4696; 2017). Taking these studies together with our data, it suggests that 1:1000), anti-pAkt (S473) (4060; 1:1000), and anti-ppERK (4370; PI4P content at the PM and Golgi regulate K-Ras trafficking via 1:3000) antibodies for immunoblotting were from Cell Signaling Technology. Goat anti-mouse IgG (G21040; 1:2000) and anti-rabbit IgG molecular mechanisms that are different but inter-related. secondary antibodies (G21234; 1:5000) were purchased from Invitrogen. In summary, we demonstrate that PI4P depletion at the Golgi Molecular Probes CellLight Mitochondria–RFP BacMam 2.0 (C10601), redistributes PtdSer from the PM to mitochondria and other MitoTracker Red FM (M2225), and sodium pyruvate (11-360-070) were endomembranes, resulting in K-Ras translocation to mitochondria. from Invitrogen. Dextrose (BDH9230) was from VWR. pLV-mitoDsRed Our study proposes newly discovered roles for Golgi-localized PI4P was deposited by Pantelis Tsoulfas (Addgene plasmid #44386) (Kitay et al., in regulating cellular trafficking of PtdSer and K-Ras. 2013). pSpCas9(BB)-2A-Puro(PX459) V2.0 was deposited by Feng Zhang (Addgene plasmid #62988). GFP-P4M-SidM was deposited by Tamas Balla (Addgene plasmid #51469). pDONR223-PI4KB was deposited by MATERIALS AND METHODS William Hahn and David Root (Addgene plasmid #23839). GFP–evectin- Plasmids and reagents 2-PH was a gift from Tomohiko Taguchi (Tohoku University, Japan). Staurosporine (STS) (BIA-S1086) was purchased from BioAustralis. Human phosphatidylserine decarboxylase (PISD) cDNA was purchased

Antibodies for detecting PI4KB (13247-1-AP; 1:1000), β-actin (60008- from GenScript (OHu25313D). Journal of Cell Science

11 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs231886. doi:10.1242/jcs.231886

Cell lines Glucose consumption measurement Madin–Darby canine kidney [MDCK, ATCC (CCL-34)], human embryonic On day 1, 2×105 MDCK cells were seeded in complete growth medium in a kidney [HEK293T, ATCC (CRL-11268)], and baby hamster kidney [BHK, 12-well plate. On day 2, cells were incubated with fresh complete growth ATCC (CCL-10)] cells were maintained in Dulbecco’s modified Eagle’s medium. On day 4, cells were incubated with fresh complete growth medium (DMEM) (10569-010, Invitrogen) or glucose/pyruvate-free medium for 2 h. The culture medium harvested before and after the DMEM (11966-025, Invitrogen). DMEM was supplemented with 2 mM incubation was analyzed using a Glucose Colorimetric Detection Kit L-glutamine (CA009-010, GenDepot), and 10% FBS (16000044, Life (EIAGLUC, Invitrogen) as per the manufacturer’s protocol, and the Technologies). All cell lines were grown at 37°C in 5% CO2. All cell lines numbers of cells per well were counted. In brief, medium sample was mixed have been frequently tested for mycoplasma using MycoAlert Mycoplasma with 1× HRP solution, substrate, and 1× glucose oxidase in wells of a clear Detection Kit (Lonza, LT07-318). half-area 96-well plate and incubated at room temp for 30 min. Absorbance was read at 560 nm. Results were normalized to number of cells. Growth medium treatment On day 1, 3×105 MDCK cells co-expressing ATP5G1(1–67)–RFP and Cellular ATP measurement mGFP–K-RasG12V or mGFP–LactC2 were seeded onto coverslips in On day 1, 2×105 of MDCK cells were seeded in complete growth medium in complete growth medium (DMEM, 10% FBS, 2 mM L-glutamine) on a a 12-well plate. On day 2, cells were supplemented with fresh complete 12-well plate. On day 2, cells were washed twice with 1× PBS and incubated growth medium. On day 4, cells were incubated with fresh complete growth with fresh complete growth medium. On day 4, cells were incubated with medium for 2 h. Cells were washed twice with ice-cold 1× PBS, and complete growth medium, glucose/pyruvate-free DMEM with 10% FBS and harvested in 1× lysis buffer (25 mM Tris-phosphate pH 7.8, 2 mM DTT, 2 mM L-glutamine [(−)glucose/pyruvate], glucose/pyruvate-free DMEM 2 mM DCTA, 10% glycerol and 1% Triton X-100). Samples were with 25 mM dextrose, 10% FBS and 2 mM L-glutamine [(−)pyruvate], microcentrifuged at 12,000 g for 5 min at 4°C and the supernatant was glucose/pyruvate-free DMEM with 1 mM sodium pyruvate, 10% FBS and collected, followed by determining protein concentrations by BCA assay. 2 mM L-glutamine [(−)glucose], or glucose/pyruvate-free DMEM with Samples were diluted 1:20 in 1× lysis buffer for use in the ATP assay. As per 25 mM dextrose, 1 mM sodium pyruvate, 10% FBS and 2 mM L-glutamine the ATP Determination Kit manufacturer’s instructions (A22066, [(+)glucose/pyruvate] for indicated time points. Invitrogen), 95 µl of standard reaction solution, which contains luciferin and firefly luciferase, and 5 µl of each diluted sample was added to the wells Western blotting of an opaque white 96-well plate. The plate was incubated at room temp for Cells were washed twice with ice-cold 1× PBS. Cells were harvested in lysis 15 min and then luminescence read. Results are normalized to protein buffer B containing 50 mM Tris-Cl (pH 7.5), 75 mM NaCl, 25 mM NaF, concentration. 5 mM MgCl2, 5 mM EGTA, 1 mM DTT, 100 μM NaVO4, 1% NP-40 plus protease and phosphatase inhibitors. SDS-PAGE and immunoblotting were Cellular ADP/ATP ratio measurement generally performed using 20 μg of lysate from each sample group. Signals On day 1, 2×105 of MDCK cells were seeded in complete growth medium in were detected by enhanced chemiluminescence (PI34578, Thermo Fisher a 12-well plate. On day 2, cells were supplemented with fresh complete Scientific) and imaged using an Amersham Imager 600 (GE Healthcare). growth medium. On day 4, cells were incubated with fresh complete growth ImageJ software (v1.51k) was used to quantitate band intensity. medium for 2 h. ADP/ATP ratio was measured using the EnzyLight ADP/ ATP Ratio Assay Kit (75878-114, Bioassay Systems) as per the Annexin V binding assay manufacturer’s instructions. In brief, cells were washed twice with ice- MDCK cells were incubated with FITC-conjugated Annexin V cold 1× PBS and harvested in 1× lysis buffer (25 mM Tris-phosphate (BMS500FI, Invitrogen) according to the manufacturer’s instructions. pH 7.8, 2 mM DTT, 2 mM DCTA, 10% glycerol and 1% Triton X-100). Annexin V-positive cells were counted using a BD AccuriC6 Analyzer with Protein concentrations were measured by means of BCA assay. 10 µg a 533/30 nm filter. protein per sample was mixed with ATP reagent in a white opaque 96-well plate, and luminescence was read after 1 min [relative luminescence unit Glycolytic rate and mitochondrial respiration assays (RLU) A]. 10 min after reading RLU A, luminescence was read again (RLU On day 1, MDCK cells were seeded in 250 µl complete DMEM growth B). Immediately after reading RLU B, ADP reagent was added to each well medium per well on XF24 cell culture plates (100777-004, Agilent). and luminescence read after 1 min (RLU C). The ADP/ATP ratio was On day 2, cells were incubated with fresh complete growth medium. determined by subtracting RLU B from RLU C, then dividing by RLU A. On day 4, cells were grown to full confluence and cell culture medium was replaced with 500 µl XF medium (103575-100, Agilent) supplemented Phospholipid supplementation with 2 mM L-glutamine only. Cells were incubated in a non-CO2 37°C Phospholipids were prepared and supplemented to cells as described incubator for 45–60 min, and OCR and ECAR were measured as readouts previously (Cho et al., 2015). Briefly, brain phosphatidylserine (PtdSer; for the mitochondrial respiration and glycolytic rate, respectively, using a 830032C) and phosphatidylcholine (PtdCho; 840053C, both from Avanti XF24-3 Seahorse Analyzer with the XF24 Extracellular Flux Assay kit Polar Lipids) were dried under a vacuum in a glass vial to remove the (100850-001, Agilent). For mitochondrial respiration, measurements solvent, reconstituted in growth medium without glucose (DMEM without were taken at four different time points: (1) before injection, (2) after glucose/pyruvate, 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate) injection of oligomycin (final 1 µM) (AAJ61898MA, Alfa Aesar), using a water sonicator, and diluted to a final concentration of 10 µM. For (3) after injection of FCCP (final 0.5 µM) (NC0904863, Cayman confocal microscopy, 3.0×105 MDCK cells stably co-expressing Chemical), and (4) after injection of rotenone (final 0.5 µM) ATP5G1(1–67)–RFP with mGFP–K-RasG12V or mGFP–LactC2 were (NC0779735, Cayman Chemical) and antimycin A (final 0.5 µM) seeded onto a glass coverslip in a 12-well plate and maintained in complete (89149-958, Enzo Life Sciences). For glycolytic rate, measurements growth medium (DMDM, 10% FBS, 2 mM L-glutamine) for 72 h. The were taken at four different time points: (1) before injection, (2) after culture medium was replaced with phospholipid-containing growth medium injection of glucose (final 10 mM) (BDH9230, VWR), (3) after injection and further incubated at 37°C in 5% CO2 for indicated time points. Cells of oligomycin (final 1 µM) (AAJ61898MA, Alfa Aesar), and (4) after were fixed with 4% PFA and imaged using a confocal microscope. injection of 2-deoxy-D-glucose (final 50 mM) (AC111980010, Thermo Fisher Scientific). For both assays, results were normalized to cell number CRISPR-Cas9-mediated knockout of phosphatidylinositol as quantified by NucLight Live Cell Stain (R37605, Life Technologies) 4-kinase B and validation and cell counting using a Biotek Cytation 5 imaging reader. Raw data were A previously reported protocol was used for CRISPR-Cas9-mediated gene exported to graphs using Seahorse Wave Desktop software (Version 2.6.0, knockout (Ran et al., 2013). Briefly, single-guide (sg) RNAs were designed Agilent) and Seahorse XF Cell Mito or Glycolysis Stress Test Report against the canine PI4KB cDNA sequence (NCBI reference sequence: XM_

Generator (v 3.0.6, Agilent). 022404993.1) using Benchling algorithm (https://www.benchling.com/ Journal of Cell Science

12 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs231886. doi:10.1242/jcs.231886 crispr/). PI4KB sgRNA2 and sgRNA6 target nucleotide positions 101 Cho, K.-J., Park, J.-H., Piggott, A. M., Salim, A. A., Gorfe, A. A., Parton, R. G., and 267, respectively, in exon 1. The sequence of sgRNA2 primers are: Capon, R. J., Lacey, E. and Hancock, J. F. (2012). Staurosporines disrupt forward 5′-CACCGcttgctaggcgtcatcacag-3′ and reverse 5′-AAACctgtgatg- phosphatidylserine trafficking and mislocalize Ras proteins. J. Biol. Chem. 287, ′ ′ 43573-43584. doi:10.1074/jbc.M112.424457 acgcctagcaagC-3 . The sequence of sgRNA6 primers are: forward 5 -CA- Cho, K. J., van der Hoeven, D., Zhou, Y., Maekawa, M., Ma, X., Chen, W., Fairn, ′ ′ CCGtaggtggatcatccaggcaa-3 and reverse 5 -AAACttgcctggatgatccacctaC- G. D. and Hancock, J. F. (2015). Inhibition of acid sphingomyelinase depletes 3′ (uppercase letters depict overhangings compatible with a Bbs1 sticky cellular phosphatidylserine and mislocalizes K-Ras from the plasma membrane. end). sgRNA2 and sgRNA6 were cloned into a CRISPR-Cas9 vector, Mol. Cell. Biol. 36, 363-374. doi:10.1128/MCB.00719-15 pSpCas9(BB)-2A-Puro (PX459). MDCK cells stably expressing mGFP–K- Cho, K.-J., Casteel, D. E., Prakash, P., Tan, L., van der Hoeven, D., Salim, A. A., RasG12V or mGFP–LactC2 were transfected with the plasmid and Kim, C., Capon, R. J., Lacey, E., Cunha, S. R. et al. (2016). AMPK and endothelial nitric oxide synthase signaling regulates K-Ras plasma membrane monoclonal selection was performed using 2 µg/ml puromycin. After interactions via cyclic GMP-dependent protein kinase 2. Mol. Cell. Biol. 36, selection, cells were cultured in 1 µg/ml puromycin. Cell lysates were 3086-3099. doi:10.1128/MCB.00365-16 prepared and immunoblotted to validate PI4KB knockout. An antibody Choy, E., Chiu, V. K., Silletti, J., Feoktistov, M., Morimoto, T., Michaelson, D., against human PI4KB amino acid residues 449–801 was used to detect Ivanov, I. E. and Philips, M. R. (1999). Endomembrane trafficking of ras: the endogenous PI4KB. For the immunoblot assay, cell lysates from HEK293T CAAX motif targets proteins to the ER and Golgi. Cell 98, 69-80. doi:10.1016/ cells overexpressing untagged human PI4KB cDNA were used as a S0092-8674(00)80607-8 positive control for the antibody. To verify CRISPR-Cas9-mediated Chung, J., Torta, F., Masai, K., Lucast, L., Czapla, H., Tanner, L. B., Narayanaswamy, P., Wenk, M. R., Nakatsu, F. and De Camilli, P. (2015). mutation, cDNA was generated from the PI4KB knockout MDCK cells Intracellular transport. PI4P/phosphatidylserine countertransport at ORP5- and stably expressing mGFP–K-RasG12V and amplified by PCR using a ORP8-mediated ER-plasma membrane contacts. 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Direct regulation Conceptualization: K.-J.C.; Methodology: K.-J.C.; Validation: T.E.M., K.M.H., of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. K.-J.C.; Formal analysis: T.E.M., M.H., K.-J.C.; Investigation: T.E.M., K.M.H., Science 275, 665-668. doi:10.1126/science.275.5300.665 A.T.S., K.-J.C.; Resources: M.H., R.S., S.M.H., K.-J.C.; Writing - original draft: Garcia, P., Gupta, R., Shah, S., Morris, A. J., Rudge, S. A., Scarlata, S., Petrova, T.E.M., K.-J.C.; Visualization: K.-J.C.; Supervision: K.-J.C.; Funding acquisition: V., McLaughlin, S. and Rebecchi, M. J. (1995). The pleckstrin homology domain K.-J.C. of phospholipase C-delta 1 binds with high affinity to phosphatidylinositol 4,5-bisphosphate in bilayer membranes. Biochemistry 34, 16228-16234. doi:10. Funding 1021/bi00049a039 This work was supported by the National Cancer Institute [R00-CA188593 to Gulyás, G., Radvánszki, G., Matuska, R., Balla, A., Hunyady, L., Balla, T. 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