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Rapid affinity purification of intracellular organelles using a twinstrep tagJian Xiong1,2,*, Jingquan He1,*, Wendy P. Xie1, Ezekiel Hinojosa1, Chandra Shekar R. Ambati3,Nagireddy Putluri3,4, Hyun-Eui Kim1,2, Michael X. Zhu1,2,‡ and Guangwei Du1,2,‡

ABSTRACTCells are internally organized into compartmentalized organelles thatexecute specialized functions. To understand the functions ofindividual organelles and their regulations, it is critical to resolve thecompositions of individual organelles, which relies on a rapid andefficient isolationmethod for specific organellar populations. Here, weintroduce a robust affinity purification method for rapid isolation ofintracellular organelles (e.g. lysosomes, mitochondria andperoxisomes) by taking advantage of the extraordinarily high affinitybetween the twin strep tag and streptavidin variants. With thismethod, we can isolate desired organelles with high purity and yield in3 min from the post-nuclear supernatant of mammalian cells or lessthan 8 min for the whole purification process. Using lysosomes as anexample, we show that the rapid procedure is especially useful forstudying transient and fast cellular activities, such as organelle-initiated signaling and organellar contents of small-molecularmetabolites. Therefore, our method offers a powerful tool to dissectspatiotemporal regulation and functions of intracellular organelles.

KEY WORDS: Twin strep tag, Lysosomes, Mitochondria,Peroxisomes

INTRODUCTIONEukaryotic cells are compartmentalized into distinct membrane-enclosed organelles. Each organelle carries hundreds to thousands ofproteins, lipids and metabolites, and performs a unique set of cellularfunctions. For example, lysosomes are the major degradationcompartments responsible for the clearance of unnecessarymaterials inside the cells (Kurz et al., 2008). Mitochondria are themajor organelles for the ATP generation required to support cellularsynthetic pathways (Chandel, 2014). Peroxisomes are the primaryvesicles that catabolize long-chain fatty acids and regulate the balanceof oxidization (Lodhi and Semenkovich, 2014; Smith and Aitchison,2013). In addition to their well-known classic functions, recentstudies have also revealed that some organelles are directly involvedin cell signaling. For example, mechanistic target of rapamycin

complex 1 (mTORC1) is recruited to and activated on the lysosomalsurface by sensing the abundance of nutrients in the lumen, such asamino acids and cholesterol (Castellano et al., 2017; Zoncu et al.,2011). Similarly, mitochondria can also function as a signalingorganelle (Chandel, 2014). For example, cytochrome c released fromthe mitochondria initiates cell death (Bhola and Letai, 2016; Burke,2017; Liu et al., 1996). Another example is AKAP family proteins,which anchor and regulate the activities of protein kinase A and othersignaling enzymes on the outer membrane of mitochondria (Chandel,2014; Esseltine and Scott, 2013).

With rapid technical advancements, profiling the global levels ofRNA, protein, lipids and metabolites has become common incurrent biomedical research. However, most of these large-scaleprofiling studies do not provide spatial information (Uhlen et al.,2015), thus cannot explain how different organelles regulate theirhighly compartmentalized cellular functions. The ability ofmeasuring the compositions of specific organellar populationsand their changes in response to stimuli would provide a powerfultool to understand the functions of these organelles.

Isolation of different organelles is traditionally accomplished bysubcellular fractionation with differential centrifugation or multi-step density gradient ultracentrifugation (de Araujo and Huber,2007; Foster et al., 2006; Frezza et al., 2007; Graham, 2001a,b,c;Michelsen and von Hagen, 2009). However, most subcellularfractionation approaches bear some intrinsic drawbacks. Forexample, the heterogeneous nature in the density of any givenorganellar population makes it difficult to obtain a type oforganelle without contamination from the others. In addition, theconcentration of a desired organellar population collected frommultiple fractions is often relatively low, making some downstreamanalyses challenging. Moreover, to the best of our knowledge, thesubcellular fractionation methods usually take more than an hour(Frezza et al., 2007; Graham, 2001a,b,c), which may lead tochanges in the compositions of organelles, especially the signalingmolecules associated with the cytoplasmic leaflet of the organellesand some labile small-molecule metabolites. Besides fractionation,specific methods have also been developed for the purification ofcertain organelles. For example, lysosomes can be isolated bymagnets after being loaded with iron oxide-conjugated dextrans(Rofe and Pryor, 2016). However, depending on the duration ofloading and chasing, dextrans are enriched to different degrees invarious endosome populations and lysosomes (Humphries et al.,2011). Moreover, long-term accumulation of non-degradabledextran may have some unexpected effects on lysosomalfunctions (Kurz et al., 2008). Some recent studies have shown thesuccessful purification of mitochondria and lysosomes byusing beads conjugated to antibodies against an endogenousmitochondrial or lysosomal-resident protein (Franko et al., 2013;Michelsen and von Hagen, 2009), or against an epitope tag fused tothese resident proteins (Abu-Remaileh et al., 2017; Ahier et al.,Received 17 June 2019; Accepted 15 November 2019

1Department of Integrative Biology and Pharmacology, McGovern Medical School,The University of Texas Health Science Center at Houston, Houston, TX 77030,USA. 2Biochemistry and Cell Biology Program, MD Anderson Cancer CenterUTHealth Graduate School of Biomedical Sciences, Houston, TX 77030, USA. 3DanL. Duncan Cancer Center, Advanced Technology Core, Alkek Center for MolecularDiscovery, Baylor College of Medicine, Houston, TX 77030, USA. 4Department ofMolecular & Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA.*These authors contributed equally to this work

‡Authors for correspondence (Michael.X.Zhu@uth.tmc.edu; guangwei.du@uth.tmc.edu)

J.H., 0000-0002-9341-8750; W.P.X., 0000-0002-0085-4415; M.X.Z., 0000-0002-5676-841X; G.D., 0000-0003-4193-6975

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© 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs235390. doi:10.1242/jcs.235390

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2018; Chen et al., 2016; Zoncu et al., 2011). Antibody affinitypurification is fast and specific; thus it overcomes some drawbacksof the traditional approaches. However, antibody affinitypurification requires a substantial amount of antibody. In addition,the elution of functional organelles is achieved via the competitionby a high concentration of epitope peptides, which is usually notvery efficient.One popular protein purification strategy is fusing the proteins of

interest to the Strep II tag (WSHPQFEK), which mimics the stronginteraction between biotin and streptavidin (Kimple et al., 2013;Schmidt and Skerra, 2007). Strep II-tagged proteins can beefficiently eluted by a low concentration of biotin derivatives. Thesmall Strep II tag is biologically inert, and the binding between thetagged proteins and streptavidin beads can take place underphysiological conditions. In addition to protein purification, thereadily reversible interaction has allowed the use of Strep II tag forthe purification of live antigen-specific T cells (Liu et al., 2016).The recently generated streptavidin variant, Strep-Tactin XT,further increases the binding affinity between two tandem Streptags (herein denoted the ‘twin strep tag’) (Schmidt et al., 2013;Yeliseev et al., 2017). As compared to micromolar-to-nanomolardissociation constant (Kd) for most commercial epitope antibodies,such as FLAG, Myc and HA (Einhauer and Jungbauer, 2001;LaCava et al., 2015; Schiweck et al., 1997; Wegner et al., 2002),twin strep tag features a nanomolar-to-picomolar Kd towards Strep-Tactin XT while preserving reversibility of binding (Yeliseev et al.,2017). In addition, streptavidin is far more stable than antibodies asit is resistant to almost any protease and to extreme pH (Bayer et al.,1990; Kimple et al., 2013), which helps to reduce the waste of beadsand obtain consistent results.In the current study, we developed a new organelle isolation

approach using the twin strep tag. We show rapid and efficientpurification of lysosomes, mitochondria and peroxisomes usingstreptavidin magnetic beads that bind to twin strep tag fused tothe cytoplasmic tail of a resident protein for lysosomes, or thetargeting signals of mitochondria and peroxisomes.

Furthermore, using lysosomes as an example, we demonstratethat this method can be used to monitor the transient lysosomalassociation of signaling protein complexes as well as small-molecule metabolites.

RESULTSDesign of a new affinity lysosome purification approachusing twin strep tagMany applications involved in organelle purification, such asevaluation of signaling events and measurement of small-moleculemetabolites, require rapid recovery as well as maintenance oforganelles in the physiological conditions during purification andelution. To test the use of twin strep tag in organelle purification, wefused the twin strep tag to the C-terminus of monomeric GFP(mGFP)-fused LAMP1, a lysosome-resident protein (hereafterdenoted Lyso-2Strep) (Fig. 1A). The cytoplasmic orientation ofthe twin strep would allow subsequent purification of lysosomesusing streptavidin beads (Fig. 1B). All lysosomal membraneproteins are synthesized in the rough endoplasmic reticulum andtransported to trans-Golgi network before they are delivered tolysosomes (Braulke and Bonifacino, 2009). The expression of Lyso-2Strep is under the control of a tetracycline-inducible promoter,which offers the option of turning off the transcription a few hoursbefore lysosome isolation. This allows lysosomal delivery of newlytranslated Lyso-2Strep protein, ensuring that the majority ofmolecules are delivered to lysosomes. The inclusion of theinducible promotor also allows the control of the expression levelof Lyso-2Strep, thus minimalizing unexpected effects caused byoverexpression of the exogenous Lyso-2Strep. We generated stableHeLa cells that inducibly express Lyso-2Strep in response todoxycycline after lentiviral infection and puromycin selection.Lyso-2Strep correctly localized to lysosomes, as demonstrated byits colocalization with the lysosomal marker LAMP2 (Fig. 1C). Thisindicates that addition of the twin strep tag does not interfere withlysosomal targeting of LAMP1 and Lyso-2Strep can be used foraffinity purification of lysosomes.

Fig. 1. Design of Lyso-2Strep for affinity purification oflysosomes. (A) Schematic diagram of Lyso-2Strep. Theexpression of the fusion protein, LAMP1–mGFP–twin strep(Lyso-2Strep), is under the control of a tetracycline-induciblepromoter. (B) Workflow of organelle purification with Lyso-2Strep. Cells expressing Lyso-2Strep are rapidly harvestedand homogenized. Post-nuclear supernatant (PNS) isincubated with streptavidin magnetic beads for a short periodof time and analyzed after three washes. (C) Fluorescenceimages of the GFP signal of Lyso-2Strep (green) andimmunofluorescence staining of LAMP2, a lysosomalmarker (red), of HeLa cells stably expressing Lyso-2Strep.Lyso-2Strep expression was induced by the addition of1 µg/ml doxycycline to the cell culture 1 day beforeimmunostaining and imaging. Scale bar: 20 μm.

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Determination of the critical factors for lysosomepurification using twin strep tagWe started to perform lysosome purification using the magneticStrep-Tactin beads from IBA Lifesciences (cat. #6-5510-050).However, the recovery of lysosomes was extremely low. Werecognized that the beads we used had a diameter of 25 μm, acommonly used size for protein purification. We reasoned that thesize of the beads might affect the binding capacity of lysosomes, andtherefore tested the efficiency of lysosome isolation usingstreptavidin beads of different sizes (i.e. 50 nm, 1 μm, 5 μm, and25 μm diameters). To avoid variations in the bead materials, thequality of streptavidin and conjugation efficiency, we purchasedthese beads from the same company (Creative Diagnostics). Weincubated equal amounts of the post-nuclear supernatant (PNS)with beads of the same binding capacity but different sizes. Wefound that the 1 μm diameter beads recovered the most lysosomes,while other beads of either larger or smaller sizes exhibitedsignificantly lower lysosomal recovery (Fig. 2A). This resultsuggests that beads with a diameter of ∼1 μm are critical to theefficient recovery of lysosomes using affinity purification.By using magnetic beads from a different source (Thermo Fisher

Scientific), we confirmed that beads with 1 μm diameter worked

markedly better than those with 25 μm size (Fig. 2B). To test thepurity of the isolated lysosomes, we performed western blot analysisfor protein levels of several organelle markers (Fig. 2C). We detectedLAMP1 and LAMP2 in the purified lysosomes, but not markers formitochondria (SDHA and TOM20), endoplasmic reticulum [SERCA(detecting SERCA1/2/3, also known as ATP2A1, ATP2A2, andATP2A3) and PDI (also known as H4BP)], Golgi [Golgin-97 (alsoknown as GOLGA1) and GOLM1], peroxisomes (catalase andSLC27A2), plasma membrane [PMCA2 and ATP1A1 (sodium/potassium-transporting ATPase subunit alpha-1)], and cytosol [S6K,and ERK1 and ERK2 (ERK1/2, also known as MAPK3 andMAPK1, respectively)] (Fig. 2C), indicating no contamination byother organelles.

To test whether the purified lysosomes remain intact, we elutedthe bound products from the streptavidin beads with biotin and thenstained them with LysoTracker, which selectively labels acidicorganelles (Chazotte, 2011a; Xiong and Zhu, 2016). The vastmajority of the purified lysosomes were labeled by the LysoTracker,indicating that they remained intact and maintained a low luminalpH (Fig. 2D). Therefore, the affinity purification of lysosomes usingLyso-2Strep is a rapid, effective and specific approach thatmaintains the intactness of the organelles.

Fig. 2. Determination of factors critical to lysosome purification. (A) Effect of bead sizes using beads from Creative Diagnostics. The same amount ofPNS was incubated with streptavidin beads of different diameters as indicated for 5 min. The volume of the beads for each size was adjusted to have the samebinding capacity. Lysosome abundance in the purified products was determined by western blotting for LAMP2, showing that 1 µm diameter beads are the mostefficient. (B) Comparison of the effect of bead sizes on lysosomal purification efficiency using beads from Thermo Fisher Scientific. Western blot analyses ofLAMP2 from PNS and products of purification of Lyso-2Strep using 25 µm and 1 µm streptavidin beads. (C) Purity of the lysosome preparation. Western blotanalyses of PNS and products after purification with Lyso-2Strep using 1 µm streptavidin beads. Intracellular organelle markers used were: LAMP1 and LAMP2(lysosomes, Lyso), SDHA and TOM20 (mitochondria, Mito), SLC27A2 and catalase (peroxisomes, Pex), PDI and SERCA (endoplasmic reticulum, ER),Golgin-97 and GOLM1 (Golgi), ATP1A1 and PMCA2 (plasmamembrane, PM), S6K and ERK1/2 (cytosol, Cyt). The purified lysosomes are free of contaminationfrom other organelles. (D) Intactness of the lysosome preparation. Fluorescence images of lysosomes isolated from HeLa cells expressing Lyso-2Strep (green),eluted from streptavidin beads with 20 mM biotin and then labeled with LysoTracker (red). Scale bar: 20 μm. (E) Effect of incubation time and bead abundance.The same amount of PNS (100 µl) was incubated with 30 µl or 60 µl of 1 µm diameter streptavidin beads (Thermo Fisher Scientific) for different time periodsas indicated. The relative recovery efficiency of lysosomes was determined by comparing the density of LAMP2 to that of PNS on the same western blots.

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We then examined how incubation time and the amount ofstreptavidin beads affect lysosome recovery efficiency. Startingfrom the same amount of PNS (100 µl), the incubation with 30 µlbeads for 2 min resulted in a yield of ∼60% of total lysosomes(Fig. 2E). Prolonging the incubation to up to 30 min did notobviously increase the recovery of lysosomes any further (Fig. 2E),suggesting that the binding capacity of the beads approachedsaturation very rapidly. The incubation time could be furthershortened by using 60 µl beads, leading to a maximal recovery oflysosomes (60–70%) in as short as 30 s (Fig. 2E). The total isolationprocedure, including cell collection and homogenization (<5 min),binding (0.5 min) and washes (2 min), could be finished in less than8 min (Fig. 1B). These results demonstrate that Lyso-2Strep allowsfor exceedingly rapid isolation of lysosomes, a feature that can beextremely critical to some analyses, such as for analyzing proteinsbound to lysosomal surfaces and small-molecule metabolites in thelysosomal lumen.

Examination of nutrient-dependent regulation of thesubcellular location of mTORC1 using purified lysosomesRecent studies have suggested that the lysosomal surface can be aplatform for some important signaling pathways, such as themTORC1 and TFEB pathways (Settembre et al., 2012; Zoncu et al.,2011). mTORC1 is the master regulator of cell metabolism and hasbeen demonstrated to be transiently activated on the surface oflysosomes in responses to the increased levels of intracellularnutrients, such as amino acids (Xiong and Zhu, 2016; Zoncu et al.,2011). Since nutrient-regulated mTORC1 association withlysosomes is transient and involves only a minor fraction of thetotal cellular pool, directly analyzing the mTORC1 complex on thelysosomes rather than that in whole cells would offer insights intomTORC1 activation and regulation. We tested whether the purifiedlysosomes by Lyso-2Strep can capture the changes of the mTORC1complex on lysosomes in HeLa cells cultured in normal, aminoacid-deprived, and amino acid deprived and then refed growthmedia. Comparing to the normal culture medium, amino acidstarvation led to an ∼60% decrease of the lysosome-associatedmTORC1 components, mTOR and Raptor, which was restored by

refeeding cells with amino acids (Fig. 3). Consistent with thechanges in lysosomal association of the mTORC1 components,the level of mTOR phosphorylation at Ser2448, which indicates theactivation status of mTORC1, on the isolated lysosomes, was alsodecreased by 60% after the 1 h amino acid starvation. Similarly, thedecrease in mTOR phosphorylation was restored by amino acidreplenishment (Fig. 3). These changes in mTORC1 on the isolatedlysosomes are consistent with previous findings using a differentapproach of lysosome isolation (Zoncu et al., 2011). Therefore,lysosome isolation using Lyso-2Strep offers an extremely powerfulapproach to studymTORC1 and other signaling events occurring onthe cytoplasmic leaflet of lysosomes.

Mass spectrometry analysis of lysosomal luminal aminoacids using isolated lysosomesRapid isolation of lysosomes would allow us to analyze metabolicactivity and metabolites in the lysosomal lumen. Lysosomes aredirectly involved in the metabolism of several macromolecules andtheir building blocks, such as proteins and amino acids. Metabolicactivity in the cytosol is subject to regulation by the levels of aminoacids in the lysosomal lumen through modulating the activity of keymetabolism regulatory proteins, such as mTORC1 (Abu-Remailehet al., 2017; Zoncu et al., 2011). As a proof of principle, wemeasured the levels of different amino acids in isolated lysosomesby mass spectrometry and compared them with those in cytosol. All20 amino acids were detected in both lysosomes and cytosol.Among them, leucine and isoleucine constitute ∼50% of totalamino acids in both cytosol and lysosomes. In contrast, theproportions of some amino acids are different between lysosomesand cytosol. For example, cysteine, arginine, aspartic acid andvaline display markedly higher proportions in lysosomes than incytosol, while phenylalanine, the second most abundant amino acidspecies, is ∼2-fold enriched in cytosol as compared to lysosomes.All other amino acid species exhibit similar proportions in cytosoland lysosomes (Fig. 4A–C). These findings suggest that undernormal culture conditions, lysosomes in HeLa cells preferentiallymaintain a limited group of amino acid species, each representingpolar, positively charged, negatively charged and neutral aminoacids, respectively. The functional significance and underlyingmechanism(s) warrant further investigation. These results highlightthe importance of analyzing individual amino acid species and othermetabolites in isolated lysosomes. Varying the metabolic conditionsof the cells before lysosome purification will likely yield crucialinsights into the mechanisms of regulation of cell metabolism.

Affinity purification of mitochondria and peroxisomes usingtwin strep tagWe also tested whether the same design can be used to purify otherorganelles, such as mitochondria and peroxisomes. Similar to whatwe undertook for Lyso-2Strep, we fused mGFP and the twin streptag to the C-terminus of the mitochondrion-targeting sequence ofthe mitochondrial resident protein TOM20 and the peroxisome-targeting sequence of the peroxisomal-resident protein PEX-3(Kapitein et al., 2010; Komatsu et al., 2010), to generate Mito-2Strep and Pex-2Strep for the purification of mitochondria andperoxisomes, respectively (Fig. 5A). HeLa cells stably expressingMito-2Strep or Pex-2Strep were established by lentiviral infectionfollowed by puromycin selection. After doxycycline induction forexpression, both Mito-2Strep and Pex-2Strep showed intendedlocalizations to mitochondria and peroxisomes, respectively, asdemonstrated by their colocalization with the mitochondrion-specific dye MitoTracker and the peroxisomal-resident protein

Fig. 3. Demonstration of mTORC1 activation on lysosomes usinglysosomes isolated with Lyso-2Strep. Western blot analysis of mTOR,phosphorylated (p)-mTOR (S2448), LAMP2 andRaptor in the PNS (Input) andpurified lysosomes (Lyso-Prep) of HeLa cells cultured in normal growthmedium (Fed), amino acid-free medium for 1 h (Starved), or amino acid-freemedium for 1 h followed by the normal growth medium for another hour(Refed). Note the decreases inmTOR, p-mTOR andRaptor in the Lyso-Prep ofthe starved samples. Representatives of three independent experiments withsimilar results.

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catalase (Fig. 5B,C). Furthermore, the isolated mitochondria werestained with MitoTracker Red or Tetramethylrhodamine ethyl ester(TMRE) (Fig. 5D,E). Given that the staining of MitoTracker Redand TMRE by mitochondria depends on mitochondrial membranepotential (Chazotte, 2011b), these results suggest that the purifiedmitochondria are intact. Importantly, TMRE staining of purifiedmitochondria was drastically reduced by a 30-min pretreatment with50 µM CCCP, a mitochondrial oxidative phosphorylationuncoupler (Fig. 5E), suggesting that the purified mitochondriapreserve the membrane potential.We then checked the purity of the isolated mitochondria and

peroxisomes by examining the presence of different organellarmarkers through western blotting. The isolated mitochondria andperoxisomes were free from the markers for cytosol and most otherorganelles except for the presence of some of the peroxisomalmarker catalase (Fig. 6A) in mitochondria and the mitochondrialmarkers SDHA and TOM20 in peroxisomes (Fig. 6B). In contrast,another peroxisomal marker, very long-chain acyl-CoA synthetase(SLC27A2), was not detected in the purified mitochondria(Fig. 6A). The co-existence of mitochondrial and peroxisomalmarkers has also been reported before in mitochondria andperoxisomes isolated by subcellular fractionation (Sugiura et al.,2017) and affinity purification methods (Bayraktar et al., 2019;Chen et al., 2017). This may reflect the intrinsic interacting nature ofthe two organellar types, instead of unspecific contamination, forthe following three reasons: (1) mitochondria and peroxisomes arephysically tethered at their contact sites (Shai et al., 2016); (2) newperoxisomes are partially derived from mitochondria and therefore

mitochondria and peroxisomes carry some common proteins(Sugiura et al., 2017); (3) SLC27A2 is absent in the purifiedmitochondria (Fig. 6A). Peroxisomal SLC27A2 is likely deriveddirectly from endoplasmic reticulum because it was previouslyshown to localize to peroxisomes and endoplasmic reticulum butnot mitochondria (Singh and Poulos, 1988; Steinberg et al., 1999).

As for the lysosome purification, we compared the capabilities ofdifferent sizes of streptavidin beads in the isolation of mitochondriaand peroxisomes. Among the different bead diameters tested,including 50 nm, 1 μm, 5 μm, and 25 μm, the 1 μm diameter beadsagain exhibited the best performance in purifying both mitochondria(Fig. 6C) and peroxisomes (Fig. 6D). Under our experimentalconditions, ∼60% of mitochondria and ∼75% of peroxisomes wererecovered from the PNS (Fig. 6E,F) within 30 s of bead incubation.Doubling the amount of beads slightly increased the recovery ofmitochondria during the shortest incubation time (30 s) withoutdramatically affecting that from longer (>1 min) time incubation(Fig. 6E,F).

DISCUSSIONA key to isolating organelles is to enrich them with high purity in ashort period of time, so that molecules associated with or inside theorganelles, especially signaling proteins and labile metabolites, canremain unchanged during purification. In the current study, wereport a general strategy for organelle purification using the twinstrep tag. The strong and specific interaction between the strep tagand streptavidin offers some distinct advantages. Under ourexperimental conditions, we can recover ∼60% of total lysosomes

Fig. 4. Measurement of amino acid contents in the cytosol and lysosomes by LC-MS. Cytosol and lysosome preparations were made from HeLa cellsstably expressing Lyso-2Strep as described in the Materials and Methods. Amino acid levels were determined by LC-MS. (A,B) Pie chart presentations ofproportions of individual amino acids in cytosol (A) and lysosomes (B). (C) Same data as in A and B plotted as a bar graph showing comparisons between cytosoland lysosomes for the proportions of individual amino acids as means±s.d. of triplicate measurements. Note: a logarithmic scale and two breaks areused to accommodate the large span of data values between aspartic acid (0.01% in cytosol) and isoleucine/leucine (∼50% in cytosol).

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and mitochondria, and more than 75% of peroxisomes, with as shortas a half minute incubation of PNS and the beads, which iscomparatively shorter than the 3.5 min incubation required forantibody-based affinity purification using 3×HA beads (Chen et al.,2017). Another advantage of our approach is that the number of cellscan be adjusted easily based on the need of subsequent application.For example, lysosomes purified from three million cells aresufficient for the western blot analyses of mTOR signalingcomponents presented in Fig. 3 and 20 million cells are sufficientfor lysosomal amino acids measurement shown in Fig. 4. Incontrast, most density gradient ultracentrifugation methods requiretypically 200–500 million cells and a time duration of several hours(de Araujo and Huber, 2007; Michelsen and von Hagen, 2009).Furthermore, ourmethod requiresminimal equipment and experience,and can be performed in any biomedical laboratory. Finally, the twinstrep purification is expected to cost less than antibody-basedapproaches, due to the high yield of organelle recovery shown inour study (thus fewer beads are needed) and excellent stability ofstreptavidin (Bayer et al., 1990; Kimple et al., 2013).The method described in the current study can be of great use in

understanding the structures and functions of intracellularorganelles. First, it is known that there are quantitative andqualitative differences in protein levels in the same type oforganelles from different cell types (Itzhak et al., 2017). Thesimplified procedures and reduced requirement for cell numbers inour approach make it possible to isolate and profile the compositesof proteins, lipids and small molecule metabolites in parallel and inthe same organellar population from different cell types. This kindof approach might be extremely important for cancer studies. Thereare overwhelmingly increasing collections of information on

genomic, transcriptional and global proteomic alterations incancer cells. However, how oncogenic alternations affect thecompositions of intracellular organelles and how these changesrelate to their specialized functions remain mysterious. Second, theability to rapidly recover the desired organelles using the currentmethod is essential for studying signal transduction on organellarsurfaces and metabolism of labile metabolites within the organelles,which either co-exist with the organelle transiently or have fastturnover rates. Using lysosomes as an example, we demonstrated thefeasibility of monitoring changes in mTORC1 signaling in responseto nutrient (amino acid) availability and of quantifying the contentsof individual amino acid species (Figs 3 and 4). Third, thegenetically encoded twin strep tag can also be used in animals toassess the physiological functions of specific organelles in vivo. Ithas been shown that mitochondria from specific cell types can bequickly isolated with the use of a mitochondrion-targeted epitopetag from complex tissues without cell sorting, which increases thespeed of isolation and allows better retention of the mitochondrialmetabolite profile (Ahier et al., 2018; Bayraktar et al., 2019). Weexpect that the tools described here can further increase thecapability of in vivo profiling of different organelles in differentcells and tissues. Finally, the twin strep tag can be used incombination with other tags, such as 3×HA, for rapid isolation ofdifferent organelles in the same cells or tissues.

While most amino acid species showed similar proportions amongthe total amino acids in the lysosomal lumen and in the cytosol in ourlysosomal amino acid measurement, a few amino acids showedpreferential lysosomal accumulations. Our overall conclusion isconsistent with a previous lysosomal amino acid measurement thatalso showed differential distribution of some amino acids in the

Fig. 5. Purification of mitochondria and peroxisomes using twin strep tag. (A) Schematic diagram of the design of Mito-2Strep and Pex-2Strep for affinitypurification of mitochondria and peroxisomes, respectively. (B) Fluorescence images of the GFP signal of Mito-2Strep (green) and MitoTracker staining ofmitochondria (red) of HeLa cells stably expressingMito-2Strep. Mito-2Strep expression was induced by the addition of 1 µg/ml doxycycline to the cell culture 1 daybefore MitoTracker staining and imaging. (C) Fluorescence images of the GFP signal of Pex-2Strep (green) and immunofluorescent staining of catalaseto label peroxisomes (red) of HeLa cells stably expressing Pex-2Strep. Pex-2Strep expression was induced by the addition of 1 µg/ml doxycycline to the cellculture one day before immunostaining and imaging. (D) MitoTracker staining of isolated mitochondria eluted from streptavidin beads with 10 mM biotin showingthe uptake of MitoTracker (red) by the purified mitochondria (green). (E) TMRE staining of isolated mitochondria and its inhibition by a 30-min pretreatment with50 µM CCCP. Mitochondria were eluted from streptavidin beads with 20 mM biotin. Scale bars: 20 µm.

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cytoplasm and lysosomal lumen (Abu-Remaileh et al., 2017).However, there are some obvious differences between the presentstudy and that of Abu-Remaileh et al. For example, the relativeproportions of cysteine, alanine, aspartate, arginine and valine aresignificantly higher in the lysosomal lumen than in cytosol in HeLacells in our study, whereas cystine is more abundant in lysosomallumen of HEK 293 cells (Abu-Remaileh et al., 2017). The differentdistributions of amino acids in the lysosomal lumen in these two celltypes may be due to different expression levels of various lysosomalamino acid transporters and different experimental conditions.However, the exact reasons remain to be further investigated.One interesting finding from our study is that the efficiency of

organelle isolation is dependent on the size of the magnetic beads.Among the beads of different diameters, 50 nm, 1 μm, 5 μm, and25 μm, tested in the current study, we found that the 1 μm diameterbeads performed the best for the isolation of lysosomes, mitochondriaand peroxisomes. This result suggests that comparable sizes betweenthe beads and organelles are critical to the success of affinitypurification of organelles, likely due to the maximal occupation (and/or exclusion) of organelles on the beads. Lysosomes, mitochondriaand perixosomes, have a diameter of 0.5–1 μm (Xu and Ren, 2015),0.75–3 μm (Wiemerslage and Lee, 2016) and 0.1–1 μm (Lodhi and

Semenkovich, 2014), respectively. It is possible that the isolationefficiency can be further increased with optimal sized beads for eachorganelle type. For experiments in which the maximal recovery isdesired, we suggest performing a pilot experiment using bead sizesbetween 10 nm and 200 μm to identify the bead size that yields thehighest recovery efficiency. It may also be possible to predict themostappropriate bead size for an organellar population by mathematicalmodeling of maximal surface interactions between the desiredorganelles and the beads. We performed most of our experimentsusing 1 μm magnetic streptavidin beads from Thermo FisherScientific. It is reported that the twin strep tag has a picomolar Kd

towards Strep-Tactin XT, a mutant form of streptavidin (Yeliseevet al., 2017). Unfortunately, except for the 50 nm sized beads,Strep-Tactin XT beads smaller than 1 μm in diameter were notcommercially available at the time of our study. We anticipate thatboth the yield and speed of purification will be further improved byStrep-Tactin XT beads of more appropriate sizes.

In summary, we describe a rapid and efficient method for theisolation of intact lysosomes, mitochondria and peroxisomes. Weprovide evidence that lysosomes purified by this method can beused to analyze signaling dynamics and metabolites. Predictably,this method should also be compatible for quick isolation of specific

Fig. 6. Characterization of isolated mitochondria and peroxisomes and determination of factors critical to the organelle purification. (A,B) Western blotanalyses for the purity of mitochondrial (A) and peroxisomal (B) preparations using intracellular organelle markers as in Fig. 2C. (C,D) Effect of bead diameter onthe isolation of mitochondria (C) and peroxisomes (D). The same amount of PNS was incubated with streptavidin beads of indicated diameters for 5 min.The volume of the beads for each size was adjusted to have the same binding capacity. Mitochondrial and peroxisomal abundances in the purified samples weredetermined by western blotting for SDHA (C) and catalase (D), respectively, showing that 1 µm diameter beads are the most efficient for both organelles.(E,F) Effect of incubation time and bead abundance on the purification of mitochondria and peroxisomes. Western blot analyses for the abundance of SDHA (E)and catalase (F) in mitochondrial and peroxisomal preparations, respectively, obtained by incubation of 100 µl PNS with 30 µl or 60 µl of 1 µm streptavidin beads(Thermo Fisher Scientific) for different time periods as indicated. The relative recovery efficiencies of mitochondria and peroxisome were determined bycomparing the densities of SDHA and catalase, respectively, in the purified organelles to that in PNS on the same Western blots.

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organellar populations from other mammalian tissues or non-mammalian cells or tissues, such as those of yeast, plant, C. elegansor Drosophila, although organism-specific organelle targetingsequence might be needed to target the twin strep fusion proteinsto the desired organelles.

MATERIALS AND METHODSGeneral reagents and antibodiesStreptavidin beads were purchased from Thermo Fisher Scientific(Waltham, MA, cat. #88817) and Creative Diagnostic (Shirley, NY;50 nm, cat. #WHM G066; 1 µm, cat. #WHM-S103; 5 µm, cat. #WHM-S108; 25 µm, cat. #WHM-S177), while Tactin beads were purchased fromIBA biosciences (Gottingen, Germany, cat. #6-5510-050). Beads werewashed with phosphate-buffered saline (PBS) before use. LysoTracker waspurchased from Thermo Fisher Scientific (Waltham, MA). Antibodies forS6K (cat. #9202, 1:1000), SDHA (cat. #5839, 1:1000), catalase (cat.#12980, 1:1000), PDI (cat. #3501, 1:1000), Golgin-97 (cat. #13192,1:1000) and ERK1/2 (cat. #4696, 1:1000) were from Cell SignalingTechnology (Danvers, MA); antibody for SERCA (cat. #SC-271669,1:1000) was from Santa Cruz Biotechnology (Dallas, TX); antibody forLAMP1 (cat. #L1418, 1:2500) was from Sigma-Aldrich (St Louis, MO);antibodies for PMCA2 (cat. #19678-1-AP, 1:1000), ATP1A1 (cat. #14418-1-AP, 1:1000), GOLM1 (15126-1-AP, 1:1000), SLC27A2 (14048-1-AP,1:1000) and TOM20 (cat. #11802-1-AP, 1:10,000) were from ProteinTech(Rosemont, IL); and antibody for LAMP2 (cat. #9840-01, 1:2500) was fromthe Developmental Studies Hybridoma Bank (Iowa City, IA). Dylight800-conjugated goat anti-mouse-IgG (cat. #SA5-10176, 1:5000) and Dylight680-conjugated goat anti-rabbit-IgG (cat. #35568, 1:5000) were fromThermoFisher Scientific. All restriction enzymes and Q5® High-Fidelity DNAPolymerase for PCR were from New England Biolabs (Ipswich, MA).

Molecular cloningLAMP1, mGFP and the twin strep tag, were amplified by PCR from LAMP-CFP-FKBP (Komatsu et al., 2010), mGFP-PASS (Lu et al., 2016; Zhanget al., 2014), and AAVS1_Puro_PGK1_3xFLAG_Twin_Strep (Addgene#68375; Dalvai et al., 2015), respectively, and fused by PCR to generate theLAMP1–mGFP–2strep fusion protein. The Lyso-strep is then cloned intoLT3G-mGFP-PASS (Lu et al., 2016) derived from L3GEPIR (Fellmannet al., 2013) that contains the third generation tetracycline-induciblepromoter, to generate LT3G–LAMP1–mGFP–2strep (Lyso-2strep). ThecDNA encoding amino acids 1–34 of TOM20 was amplified by PCR fromTOM20-CFP-FRB (Komatsu et al., 2010) and then cut with NheI andBamHI at the artificially introduced sites. To generate LT3G–Mito–mGFP–2strep (Mito-2Strep), LT3G–LAMP1–mGFP–2strep was cut with NheI andBamHI, and the LAMP1 fragment was replaced with the TOM20 fragment.The cDNA encoding amino acids 1–42 of PEX3 was amplified by PCRfrom pβactin-PEX3-mRFP (Kapitein et al., 2010) and then cut with NheIand BamHI at the artificially introduced sites. To generate LT3G–Pex–mGFP–2strep (Pex-2Strep), the LAMP1 fragment of LT3G–LAMP1–mGFP–2strep was removed by NheI and BamHI digestion, and thenreplaced with the cut PEX3 fragment. The full sequences and maps of theseconstructs will be shared upon request.

Cell culture, lentivirus production and transductionHeLa cells from ATCC (Manassas, VA) were maintained in Dulbecco’smodified Eagle’s medium (DMEM) plus 10% fetal bovine serum (FBS)(Thermo Fisher Scientific). Cells were passaged less than five passagesbefore being used for the current study. For amino acid starvation, cells weretreated with amino acid-free DMEM (US Biological) supplemented with10% FBS that has been dialyzed with Slide-A-Lyzer™ Dialysis Cassetteswith 3.5 kDa cut-off (Thermo Fisher Scientific). Lentiviruses were collectedfrom TLA-293T cells (Thermo Fisher Scientific) co-transfected with thelentiviral vector (Lyso-2Strep, Mito-2Strep, or Pex-2Strep), pCMV-dR8.2and pMD2.G using Lipofectamine and Plus reagent (Thermo FisherScientific) as described previously (He et al., 2017; Wang et al., 2017). At 2days after infection with lentiviruses, HeLa cells were selected withpuromycin (1 μg/ml) for stable expression.

Isolation of lysosomes, mitochondria and peroxisomesApproximately 6×106 HeLa cells stably expressing Lyso-2Strep, Mito-2Strep or PEX-2Strep, were seeded on a 150-mm cell culture dish overnightand 1 μg/ml of doxycycline was added at the time of seeding. On the day ofexperiment, medium was replaced with fresh medium free of doxycycline∼3 h before the experiment. All organelle isolation procedures wereperformed in a cold room with ice-cold reagents. Cells were washed twicewith PBS, collected in 1 ml of potassium phosphate-buffered saline (KPBS;136 mM KCl, 10 mM KH2PO4, pH 7.3) with a cell lifter, and thentransferred into a 1.5 ml centrifuge tube. The cells were centrifuged for1 min at 1000 g and then resuspended in 1 ml of KPBS. Resuspended cellswere homogenized in a 2 ml glass tissue grinder (VWR, Radnor, PA) with30 gentle and continuous strokes. The homogenates were centrifuged at1000 g for 2 min and 800 μl of the post-nuclear supernatant (PNS) wasadded to 150 μl or 300 μl of prewashed streptavidin-conjugated magneticbeads (cat. #88817, Thermo Fisher Scientific) in a 1.5 ml centrifuge tube.The PNS and beads were gently mixed and incubated in a tube rotator for0.5–30 min to allow binding. After incubation, the beads were collectedwith a magnetic stand (cat. #12321D, Thermo Fisher Scientific). Thesupernatant was discarded and beads resuspended in 1 ml KPBS and thentransferred to a new 1.5 ml tube. The beads were washed two more times byresuspending in 1 ml KPBS and pelleting with the magnetic stand. Forimaging, the organelles were eluted from the beads by incubation with100 μl of 20 mM biotin in KPBS for 10 min, followed by collecting thebeads with the magnetic stand and saving the supernatant.

Western blottingIsolated organelles on the beads or PNSwere mixed with an equal volume of2×SDS sampling buffer. Protein samples were separated by SDS-PAGE andtransferred onto nitrocellulose membranes. Membranes were blocked with1% casein in Tris-buffered saline (TBS, 50 mM Tris-HCl, 150 mM NaCl,pH 7.5) and probed with the indicated primary antibodies diluted in 1%casein in TBS supplemented with 0.1% Tween-20 (TBST), followed byfluorescently labeled secondary antibodies. The fluorescent signals weredetected with the Li-COR Odyssey infrared imaging system from Li-CORBiotechnology (Lincoln, NE). The signal intensities of bands were analyzedwith Image Studio Lite (Li-COR). The median value of blank space aroundeach band was set as the background and subtracted from the correspondingband intensities. Western blots are representatives of at least three biologicalreplicates from independent experiments with similar results.

Fluorescence microscopyHeLa cells expressing the organelle probes were stained with antibodiesspecific for individual organelles, and visualized with a Nikon A1 confocalmicroscope. Cells, purified lysosomes or mitochondria, were incubated with1 μM LysoTracker (Thermo Fisher Scientific), MitoTracker (Cell SignalingTechnology) or TMRE (Sigma-Aldrich) for 15 min at room temperaturebefore imaging. For CCCP treatment, after the third wash duringmitochondrial isolation, beads were resuspended in 500 µl KPBScontaining 2 mM sodium pyruvate and 50 µM CCCP and incubated atroom temperature for 30 min. TMRE was then added to the solution to afinal concentration of 1 µM and incubated at room temperature for 15 min.After incubation, beads were washed twice in 500 µl KPBS containing2 mM sodium pyruvate and 50 µM CCCP. Mitochondria not treated withCCCP were processed in parallel for the same duration and temperatureexposure. The bound mitochondria were eluted from beads in 100 µl 20 mMbiotin in KPBS containing 2 mM sodium pyruvate for 10 min at roomtemperature. Fluorescence images are representatives of at least threeindependent experiments with similar results.

Sample preparation for mass spectrometry analysisof amino acidsAfter the final wash, beads bound with lysosomes were incubated with 60 µlof 50% methanol on ice for 5 min. Beads were then collected with amagnetic stand and eluates were transferred to a clean glass vial (VWR). Forcytosolic amino acids, the cytosol was prepared by centrifugation of PNS at16,000 g for 3 min at 4°C to precipitate all organelles. Then 40 µl of thesupernatant was mixed with 40 µl 100% liquid chromatography and mass

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spectrometry (LC-MS) grade methanol. The samples for LC-MS analysiswere prepared by spiking 5 µl of isotopic labeled standard mix into eachsample.

Reagents and internal standards for mass spectrometryHigh-performance liquid chromatography (HPLC)-grade acetonitrile,methanol and water were procured from Burdick & Jackson (Morristown,NJ). Mass spectrometry-grade formic acid was purchased from Sigma-Aldrich. Calibration solution containing multiple calibrants in a solution ofacetonitrile, trifluroacetic acid and water was purchased from AgilentTechnologies (Santa Clara, CA). Metabolite standards and internalstandards, including N-acetyl aspartic acid-d3, tryptophan-15N2,sarcosine-d3, glutamic acid-d5, thymine-d4, gibberellic acid, trans-zeatine, jasmonic acid, 15N anthranilic acid and testosterone-d3, werepurchased from Sigma-Aldrich.

Identification of amino acidsAnalysis of metabolites was performed at the Metabolomics Core of BaylorCollege ofMedicine (Houston, TX). Amino acids were identified by Zorbaxeclipse XDB C-18 chromatography column (Agilent Technologies) using0.1% formic acid (buffer A) and 0.1% formic acid in acetonitrile (buffer B).The samples were analyzed on 6490 triple quadrupole mass spectrometercoupled with 1290 series HPLC system equipped with a degasser, binarypump, thermostatted auto sampler and column oven (Agilent Technologies).Data analysis was carried out by using Agilent Mass Hunter workstationsoftware. All the identified amino acids were normalized to the levels ofspiked isotope labeled standard. LC-MS analysis was performed in MRMmode. Source parameters used were as follows: gas temperature, 250°C; gasflow, 14 l/min; nebulizer gas pressure, 20 psi; sheath gas temperature, 350°C;sheath gas flow, 12 l/min; capillary voltage, 3000 V positive and 3000 Vnegative; nozzle voltage, 1500 V positive and 1500 V negative.Approximately 8–11 data points were acquired per detected amino acid.

AcknowledgementsWe thank Dr Takanari Inoue (Johns Hopkins University, Baltimore, MD) forLAMP-CFP-FKBP and TOM20-CFP-FRB plasmids, Dr Casper Hoogenraad(Utrecht University, The Netherlands) for pβactin-PEX3-mRFP plasmid andDr Johannes Zuber (Institute of Molecular Pathology, Vienna Biocenter, Austria)for L3GEPIR plasmid.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: G.D.; Methodology: J.X., J.H., W.P.X., E.H., C.S.R.A., N.P.,H.-E.K., M.X.Z., G.D.; Validation: W.P.X.; Formal analysis: J.X., J.H.; Investigation:J.X., J.H., G.D.; Resources: C.S.R.A., N.P., H.-E.K.; Data curation: J.X., M.X.Z.,G.D.; Writing - original draft: J.X., G.D.; Writing - review & editing: J.X., M.X.Z.,G.D.; Supervision: M.X.Z., G.D.; Project administration: G.D.; Funding acquisition:N.P., M.X.Z., G.D.

FundingThis study was supported in part by grants from American Heart Association(19TPA34910051 to G.D.), National Institutes of Health (AR075830 to G.D., andNS092377 to M.X.Z. and P30 CA125123 to N.P.), Cancer Prevention and ResearchInstitute of Texas (CPRIT) Proteomics and Metabolomics Core Facility Grants(RP170005 to N.P.), and Dan L. Duncan Cancer Center. Deposited in PMC forrelease after 12 months.

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