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

TOOLS AND RESOURCES Rapid affinity purification of intracellular organelles using a twin strep tag Jian 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,‡

ABSTRACT complex 1 (mTORC1) is recruited to and activated on the lysosomal Cells are internally organized into compartmentalized organelles that surface by sensing the abundance of nutrients in the lumen, such as execute specialized functions. To understand the functions of amino acids and cholesterol (Castellano et al., 2017; Zoncu et al., individual organelles and their regulations, it is critical to resolve the 2011). Similarly, mitochondria can also function as a signaling compositions of individual organelles, which relies on a rapid and organelle (Chandel, 2014). For example, cytochrome c released from efficient isolation method for specific organellar populations. Here, we the mitochondria initiates cell death (Bhola and Letai, 2016; Burke, introduce a robust affinity purification method for rapid isolation of 2017; Liu et al., 1996). Another example is AKAP family proteins, intracellular organelles (e.g. lysosomes, mitochondria and which anchor and regulate the activities of protein kinase A and other peroxisomes) by taking advantage of the extraordinarily high affinity signaling enzymes on the outer membrane of mitochondria (Chandel, between the twin strep tag and streptavidin variants. With this 2014; Esseltine and Scott, 2013). method, we can isolate desired organelles with high purity and yield in With rapid technical advancements, profiling the global levels of 3 min from the post-nuclear supernatant of mammalian cells or less RNA, protein, lipids and metabolites has become common in than 8 min for the whole purification process. Using lysosomes as an current biomedical research. However, most of these large-scale example, we show that the rapid procedure is especially useful for profiling studies do not provide spatial information (Uhlen et al., studying transient and fast cellular activities, such as organelle- 2015), thus cannot explain how different organelles regulate their initiated signaling and organellar contents of small-molecular highly compartmentalized cellular functions. The ability of metabolites. Therefore, our method offers a powerful tool to dissect measuring the compositions of specific organellar populations spatiotemporal regulation and functions of intracellular organelles. and their changes in response to stimuli would provide a powerful tool to understand the functions of these organelles. KEY WORDS: Twin strep tag, Lysosomes, Mitochondria, Isolation of different organelles is traditionally accomplished by Peroxisomes subcellular fractionation with differential centrifugation or multi- step density gradient ultracentrifugation (de Araujo and Huber, INTRODUCTION 2007; Foster et al., 2006; Frezza et al., 2007; Graham, 2001a,b,c; Eukaryotic cells are compartmentalized into distinct membrane- Michelsen and von Hagen, 2009). However, most subcellular enclosed organelles. Each organelle carries hundreds to thousands of fractionation approaches bear some intrinsic drawbacks. For proteins, lipids and metabolites, and performs a unique set of cellular example, the heterogeneous nature in the density of any given functions. For example, lysosomes are the major degradation organellar population makes it difficult to obtain a type of compartments responsible for the clearance of unnecessary organelle without contamination from the others. In addition, the materials inside the cells (Kurz et al., 2008). Mitochondria are the concentration of a desired organellar population collected from major organelles for the ATP generation required to support cellular multiple fractions is often relatively low, making some downstream synthetic pathways (Chandel, 2014). Peroxisomes are the primary analyses challenging. Moreover, to the best of our knowledge, the vesicles that catabolize long-chain fatty acids and regulate the balance subcellular fractionation methods usually take more than an hour of oxidization (Lodhi and Semenkovich, 2014; Smith and Aitchison, (Frezza et al., 2007; Graham, 2001a,b,c), which may lead to 2013). In addition to their well-known classic functions, recent changes in the compositions of organelles, especially the signaling studies have also revealed that some organelles are directly involved molecules associated with the cytoplasmic leaflet of the organelles in cell signaling. For example, mechanistic target of rapamycin and some labile small-molecule metabolites. Besides fractionation, specific methods have also been developed for the purification of certain organelles. For example, lysosomes can be isolated by 1Department of Integrative Biology and Pharmacology, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, magnets after being loaded with iron oxide-conjugated dextrans USA. 2Biochemistry and Cell Biology Program, MD Anderson Cancer Center (Rofe and Pryor, 2016). However, depending on the duration of UTHealth Graduate School of Biomedical Sciences, Houston, TX 77030, USA. 3Dan loading and chasing, dextrans are enriched to different degrees in L. Duncan Cancer Center, Advanced Technology Core, Alkek Center for Molecular Discovery, Baylor College of Medicine, Houston, TX 77030, USA. 4Department of various endosome populations and lysosomes (Humphries et al., Molecular & Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA. 2011). Moreover, long-term accumulation of non-degradable *These authors contributed equally to this work dextran may have some unexpected effects on lysosomal ‡Authors for correspondence ([email protected]; guangwei.du@uth. functions (Kurz et al., 2008). Some recent studies have shown the tmc.edu) successful purification of mitochondria and lysosomes by using beads conjugated to antibodies against an endogenous 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 mitochondrial or lysosomal-resident protein (Franko et al., 2013; Michelsen and von Hagen, 2009), or against an epitope tag fused to

Received 17 June 2019; Accepted 15 November 2019 these resident proteins (Abu-Remaileh et al., 2017; Ahier et al., Journal of Cell Science

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2018; Chen et al., 2016; Zoncu et al., 2011). Antibody affinity Furthermore, using lysosomes as an example, we demonstrate purification is fast and specific; thus it overcomes some drawbacks that this method can be used to monitor the transient lysosomal of the traditional approaches. However, antibody affinity association of signaling protein complexes as well as small- purification requires a substantial amount of antibody. In addition, molecule metabolites. the elution of functional organelles is achieved via the competition by a high concentration of epitope peptides, which is usually not RESULTS very efficient. Design of a new affinity lysosome purification approach One popular protein purification strategy is fusing the proteins of using twin strep tag interest to the Strep II tag (WSHPQFEK), which mimics the strong Many applications involved in organelle purification, such as interaction between biotin and streptavidin (Kimple et al., 2013; evaluation of signaling events and measurement of small-molecule Schmidt and Skerra, 2007). Strep II-tagged proteins can be metabolites, require rapid recovery as well as maintenance of efficiently eluted by a low concentration of biotin derivatives. The organelles in the physiological conditions during purification and small Strep II tag is biologically inert, and the binding between the elution. To test the use of twin strep tag in organelle purification, we tagged proteins and streptavidin beads can take place under fused the twin strep tag to the C-terminus of monomeric GFP physiological conditions. In addition to protein purification, the (mGFP)-fused LAMP1, a lysosome-resident protein (hereafter readily reversible interaction has allowed the use of Strep II tag for denoted Lyso-2Strep) (Fig. 1A). The cytoplasmic orientation of the purification of live antigen-specific T cells (Liu et al., 2016). the twin strep would allow subsequent purification of lysosomes The recently generated streptavidin variant, Strep-Tactin XT, using streptavidin beads (Fig. 1B). All lysosomal membrane further increases the binding affinity between two tandem Strep proteins are synthesized in the rough endoplasmic reticulum and tags (herein denoted the ‘twin strep tag’) (Schmidt et al., 2013; transported to trans-Golgi network before they are delivered to Yeliseev et al., 2017). As compared to micromolar-to-nanomolar lysosomes (Braulke and Bonifacino, 2009). The expression of Lyso- dissociation constant (Kd) for most commercial epitope antibodies, 2Strep is under the control of a tetracycline-inducible promoter, such as FLAG, Myc and HA (Einhauer and Jungbauer, 2001; which offers the option of turning off the transcription a few hours LaCava et al., 2015; Schiweck et al., 1997; Wegner et al., 2002), before lysosome isolation. This allows lysosomal delivery of newly twin strep tag features a nanomolar-to-picomolar Kd towards Strep- translated Lyso-2Strep protein, ensuring that the majority of Tactin XT while preserving reversibility of binding (Yeliseev et al., molecules are delivered to lysosomes. The inclusion of the 2017). In addition, streptavidin is far more stable than antibodies as inducible promotor also allows the control of the expression level it is resistant to almost any protease and to extreme pH (Bayer et al., of Lyso-2Strep, thus minimalizing unexpected effects caused by 1990; Kimple et al., 2013), which helps to reduce the waste of beads overexpression of the exogenous Lyso-2Strep. We generated stable and obtain consistent results. HeLa cells that inducibly express Lyso-2Strep in response to In the current study, we developed a new organelle isolation doxycycline after lentiviral infection and puromycin selection. approach using the twin strep tag. We show rapid and efficient Lyso-2Strep correctly localized to lysosomes, as demonstrated by purification of lysosomes, mitochondria and peroxisomes using its colocalization with the lysosomal marker LAMP2 (Fig. 1C). This streptavidin magnetic beads that bind to twin strep tag fused to indicates that addition of the twin strep tag does not interfere with the cytoplasmic tail of a resident protein for lysosomes, or the lysosomal targeting of LAMP1 and Lyso-2Strep can be used for targeting signals of mitochondria and peroxisomes. affinity purification of lysosomes.

Fig. 1. Design of Lyso-2Strep for affinity purification of lysosomes. (A) Schematic diagram of Lyso-2Strep. The expression of the fusion protein, LAMP1–mGFP–twin strep (Lyso-2Strep), is under the control of a tetracycline-inducible promoter. (B) Workflow of organelle purification with Lyso- 2Strep. Cells expressing Lyso-2Strep are rapidly harvested and homogenized. Post-nuclear supernatant (PNS) is incubated with streptavidin magnetic beads for a short period of time and analyzed after three washes. (C) Fluorescence images of the GFP signal of Lyso-2Strep (green) and immunofluorescence staining of LAMP2, a lysosomal marker (red), of HeLa cells stably expressing Lyso-2Strep. Lyso-2Strep expression was induced by the addition of 1 µg/ml doxycycline to the cell culture 1 day before immunostaining and imaging. Scale bar: 20 μm. Journal of Cell Science

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Determination of the critical factors for lysosome markedly better than those with 25 μm size (Fig. 2B). To test the purification using twin strep tag purity of the isolated lysosomes, we performed western blot analysis We started to perform lysosome purification using the magnetic for protein levels of several organelle markers (Fig. 2C). We detected Strep-Tactin beads from IBA Lifesciences (cat. #6-5510-050). LAMP1 and LAMP2 in the purified lysosomes, but not markers for However, the recovery of lysosomes was extremely low. We mitochondria (SDHA and TOM20), endoplasmic reticulum [SERCA recognized that the beads we used had a diameter of 25 μm, a (detecting SERCA1/2/3, also known as ATP2A1, ATP2A2, and commonly used size for protein purification. We reasoned that the ATP2A3) and PDI (also known as H4BP)], Golgi [Golgin-97 (also size of the beads might affect the binding capacity of lysosomes, and known as GOLGA1) and GOLM1], peroxisomes (catalase and therefore tested the efficiency of lysosome isolation using SLC27A2), plasma membrane [PMCA2 and ATP1A1 (sodium/ streptavidin beads of different sizes (i.e. 50 nm, 1 μm, 5 μm, and potassium-transporting ATPase subunit alpha-1)], and cytosol [S6K, 25 μm diameters). To avoid variations in the bead materials, the and ERK1 and ERK2 (ERK1/2, also known as MAPK3 and quality of streptavidin and conjugation efficiency, we purchased MAPK1, respectively)] (Fig. 2C), indicating no contamination by these beads from the same company (Creative Diagnostics). We other organelles. incubated equal amounts of the post-nuclear supernatant (PNS) To test whether the purified lysosomes remain intact, we eluted with beads of the same binding capacity but different sizes. We the bound products from the streptavidin beads with biotin and then found that the 1 μm diameter beads recovered the most lysosomes, stained them with LysoTracker, which selectively labels acidic while other beads of either larger or smaller sizes exhibited organelles (Chazotte, 2011a; Xiong and Zhu, 2016). The vast significantly lower lysosomal recovery (Fig. 2A). This result majority of the purified lysosomes were labeled by the LysoTracker, suggests that beads with a diameter of ∼1 μm are critical to the indicating that they remained intact and maintained a low luminal efficient recovery of lysosomes using affinity purification. pH (Fig. 2D). Therefore, the affinity purification of lysosomes using By using magnetic beads from a different source (Thermo Fisher Lyso-2Strep is a rapid, effective and specific approach that Scientific), we confirmed that beads with 1 μm diameter worked maintains 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 of PNS 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 same binding capacity. Lysosome abundance in the purified products was determined by western blotting for LAMP2, showing that 1 µm diameter beads are the most efficient. (B) Comparison of the effect of bead sizes on lysosomal purification efficiency using beads from Thermo Fisher Scientific. Western blot analyses of LAMP2 from PNS and products of purification of Lyso-2Strep using 25 µm and 1 µm streptavidin beads. (C) Purity of the lysosome preparation. Western blot analyses 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 (plasma membrane, PM), S6K and ERK1/2 (cytosol, Cyt). The purified lysosomes are free of contamination from 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 periods as indicated. The relative recovery efficiency of lysosomes was determined by comparing the density of LAMP2 to that of PNS on the same western blots. Journal of Cell Science

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We then examined how incubation time and the amount of refeeding cells with amino acids (Fig. 3). Consistent with the streptavidin beads affect lysosome recovery efficiency. Starting changes in lysosomal association of the mTORC1 components, from the same amount of PNS (100 µl), the incubation with 30 µl the level of mTOR phosphorylation at Ser2448, which indicates the beads for 2 min resulted in a yield of ∼60% of total lysosomes activation status of mTORC1, on the isolated lysosomes, was also (Fig. 2E). Prolonging the incubation to up to 30 min did not decreased by 60% after the 1 h amino acid starvation. Similarly, the obviously increase the recovery of lysosomes any further (Fig. 2E), decrease in mTOR phosphorylation was restored by amino acid suggesting that the binding capacity of the beads approached replenishment (Fig. 3). These changes in mTORC1 on the isolated saturation very rapidly. The incubation time could be further lysosomes are consistent with previous findings using a different shortened by using 60 µl beads, leading to a maximal recovery of approach of lysosome isolation (Zoncu et al., 2011). Therefore, lysosomes (60–70%) in as short as 30 s (Fig. 2E). The total isolation lysosome isolation using Lyso-2Strep offers an extremely powerful procedure, including cell collection and homogenization (<5 min), approach to study mTORC1 and other signaling events occurring on binding (0.5 min) and washes (2 min), could be finished in less than the cytoplasmic leaflet of lysosomes. 8 min (Fig. 1B). These results demonstrate that Lyso-2Strep allows for exceedingly rapid isolation of lysosomes, a feature that can be Mass spectrometry analysis of lysosomal luminal amino extremely critical to some analyses, such as for analyzing proteins acids using isolated lysosomes bound to lysosomal surfaces and small-molecule metabolites in the Rapid isolation of lysosomes would allow us to analyze metabolic lysosomal lumen. activity and metabolites in the lysosomal lumen. Lysosomes are directly involved in the metabolism of several macromolecules and Examination of nutrient-dependent regulation of the their building blocks, such as proteins and amino acids. Metabolic subcellular location of mTORC1 using purified lysosomes activity in the cytosol is subject to regulation by the levels of amino Recent studies have suggested that the lysosomal surface can be a acids in the lysosomal lumen through modulating the activity of key platform for some important signaling pathways, such as the metabolism regulatory proteins, such as mTORC1 (Abu-Remaileh mTORC1 and TFEB pathways (Settembre et al., 2012; Zoncu et al., et al., 2017; Zoncu et al., 2011). As a proof of principle, we 2011). mTORC1 is the master regulator of cell metabolism and has measured the levels of different amino acids in isolated lysosomes been demonstrated to be transiently activated on the surface of by mass spectrometry and compared them with those in cytosol. All lysosomes in responses to the increased levels of intracellular 20 amino acids were detected in both lysosomes and cytosol. nutrients, such as amino acids (Xiong and Zhu, 2016; Zoncu et al., Among them, leucine and isoleucine constitute ∼50% of total 2011). Since nutrient-regulated mTORC1 association with amino acids in both cytosol and lysosomes. In contrast, the lysosomes is transient and involves only a minor fraction of the proportions of some amino acids are different between lysosomes total cellular pool, directly analyzing the mTORC1 complex on the and cytosol. For example, cysteine, arginine, aspartic acid and lysosomes rather than that in whole cells would offer insights into valine display markedly higher proportions in lysosomes than in mTORC1 activation and regulation. We tested whether the purified cytosol, while phenylalanine, the second most abundant amino acid lysosomes by Lyso-2Strep can capture the changes of the mTORC1 species, is ∼2-fold enriched in cytosol as compared to lysosomes. complex on lysosomes in HeLa cells cultured in normal, amino All other amino acid species exhibit similar proportions in cytosol acid-deprived, and amino acid deprived and then refed growth and lysosomes (Fig. 4A–C). These findings suggest that under media. Comparing to the normal culture medium, amino acid normal culture conditions, lysosomes in HeLa cells preferentially starvation led to an ∼60% decrease of the lysosome-associated maintain a limited group of amino acid species, each representing mTORC1 components, mTOR and Raptor, which was restored by polar, positively charged, negatively charged and neutral amino acids, respectively. The functional significance and underlying mechanism(s) warrant further investigation. These results highlight the importance of analyzing individual amino acid species and other metabolites in isolated lysosomes. Varying the metabolic conditions of the cells before lysosome purification will likely yield crucial insights into the mechanisms of regulation of cell metabolism.

Affinity purification of mitochondria and peroxisomes using twin strep tag We also tested whether the same design can be used to purify other organelles, such as mitochondria and peroxisomes. Similar to what we undertook for Lyso-2Strep, we fused mGFP and the twin strep tag to the C-terminus of the mitochondrion-targeting sequence of the 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- Fig. 3. Demonstration of mTORC1 activation on lysosomes using 2Strep and Pex-2Strep for the purification of mitochondria and lysosomes isolated with Lyso-2Strep. Western blot analysis of mTOR, peroxisomes, respectively (Fig. 5A). HeLa cells stably expressing phosphorylated (p)-mTOR (S2448), LAMP2 and Raptor in the PNS (Input) and Mito-2Strep or Pex-2Strep were established by lentiviral infection purified lysosomes (Lyso-Prep) of HeLa cells cultured in normal growth followed by puromycin selection. After doxycycline induction for medium (Fed), amino acid-free medium for 1 h (Starved), or amino acid-free expression, both Mito-2Strep and Pex-2Strep showed intended medium for 1 h followed by the normal growth medium for another hour (Refed). Note the decreases in mTOR, p-mTOR and Raptor in the Lyso-Prep of localizations to mitochondria and peroxisomes, respectively, as the starved samples. Representatives of three independent experiments with demonstrated by their colocalization with the mitochondrion- similar results. specific dye MitoTracker and the peroxisomal-resident protein Journal of Cell Science

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Fig. 4. Measurement of amino acid contents in the cytosol and lysosomes by LC-MS. Cytosol and lysosome preparations were made from HeLa cells stably expressing Lyso-2Strep as described in the Materials and Methods. Amino acid levels were determined by LC-MS. (A,B) Pie chart presentations of proportions 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 cytosol and lysosomes for the proportions of individual amino acids as means±s.d. of triplicate measurements. Note: a logarithmic scale and two breaks are used to accommodate the large span of data values between aspartic acid (0.01% in cytosol) and isoleucine/leucine (∼50% in cytosol). catalase (Fig. 5B,C). Furthermore, the isolated mitochondria were mitochondria and peroxisomes carry some common proteins stained with MitoTracker Red or Tetramethylrhodamine ethyl ester (Sugiura et al., 2017); (3) SLC27A2 is absent in the purified (TMRE) (Fig. 5D,E). Given that the staining of MitoTracker Red mitochondria (Fig. 6A). Peroxisomal SLC27A2 is likely derived and TMRE by mitochondria depends on mitochondrial membrane directly from endoplasmic reticulum because it was previously potential (Chazotte, 2011b), these results suggest that the purified shown to localize to peroxisomes and endoplasmic reticulum but mitochondria are intact. Importantly, TMRE staining of purified not mitochondria (Singh and Poulos, 1988; Steinberg et al., 1999). mitochondria was drastically reduced by a 30-min pretreatment with As for the lysosome purification, we compared the capabilities of 50 µM CCCP, a mitochondrial oxidative phosphorylation different sizes of streptavidin beads in the isolation of mitochondria uncoupler (Fig. 5E), suggesting that the purified mitochondria and peroxisomes. Among the different bead diameters tested, preserve the membrane potential. including 50 nm, 1 μm, 5 μm, and 25 μm, the 1 μm diameter beads We then checked the purity of the isolated mitochondria and again exhibited the best performance in purifying both mitochondria peroxisomes by examining the presence of different organellar (Fig. 6C) and peroxisomes (Fig. 6D). Under our experimental markers through western blotting. The isolated mitochondria and conditions, ∼60% of mitochondria and ∼75% of peroxisomes were peroxisomes were free from the markers for cytosol and most other recovered from the PNS (Fig. 6E,F) within 30 s of bead incubation. organelles except for the presence of some of the peroxisomal Doubling the amount of beads slightly increased the recovery of marker catalase (Fig. 6A) in mitochondria and the mitochondrial mitochondria during the shortest incubation time (30 s) without markers SDHA and TOM20 in peroxisomes (Fig. 6B). In contrast, dramatically affecting that from longer (>1 min) time incubation another peroxisomal marker, very long-chain acyl-CoA synthetase (Fig. 6E,F). (SLC27A2), was not detected in the purified mitochondria (Fig. 6A). The co-existence of mitochondrial and peroxisomal DISCUSSION markers has also been reported before in mitochondria and A key to isolating organelles is to enrich them with high purity in a peroxisomes isolated by subcellular fractionation (Sugiura et al., short period of time, so that molecules associated with or inside the 2017) and affinity purification methods (Bayraktar et al., 2019; organelles, especially signaling proteins and labile metabolites, can Chen et al., 2017). This may reflect the intrinsic interacting nature of remain unchanged during purification. In the current study, we the two organellar types, instead of unspecific contamination, for report a general strategy for organelle purification using the twin the following three reasons: (1) mitochondria and peroxisomes are strep tag. The strong and specific interaction between the strep tag physically tethered at their contact sites (Shai et al., 2016); (2) new and streptavidin offers some distinct advantages. Under our peroxisomes are partially derived from mitochondria and therefore experimental conditions, we can recover ∼60% of total lysosomes Journal of Cell Science

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Fig. 5. Purification of mitochondria and peroxisomes using twin strep tag. (A) Schematic diagram of the design of Mito-2Strep and Pex-2Strep for affinity purification of mitochondria and peroxisomes, respectively. (B) Fluorescence images of the GFP signal of Mito-2Strep (green) and MitoTracker staining of mitochondria (red) of HeLa cells stably expressing Mito-2Strep. Mito-2Strep expression was induced by the addition of 1 µg/ml doxycycline to the cell culture 1 day before MitoTracker staining and imaging. (C) Fluorescence images of the GFP signal of Pex-2Strep (green) and immunofluorescent staining of catalase to 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 cell culture one day before immunostaining and imaging. (D) MitoTracker staining of isolated mitochondria eluted from streptavidin beads with 10 mM biotin showing the uptake of MitoTracker (red) by the purified mitochondria (green). (E) TMRE staining of isolated mitochondria and its inhibition by a 30-min pretreatment with 50 µM CCCP. Mitochondria were eluted from streptavidin beads with 20 mM biotin. Scale bars: 20 µm. and mitochondria, and more than 75% of peroxisomes, with as short genomic, transcriptional and global proteomic alterations in as a half minute incubation of PNS and the beads, which is cancer cells. However, how oncogenic alternations affect the comparatively shorter than the 3.5 min incubation required for compositions of intracellular organelles and how these changes antibody-based affinity purification using 3×HA beads (Chen et al., relate to their specialized functions remain mysterious. Second, the 2017). Another advantage of our approach is that the number of cells ability to rapidly recover the desired organelles using the current can be adjusted easily based on the need of subsequent application. method is essential for studying signal transduction on organellar For example, lysosomes purified from three million cells are surfaces and metabolism of labile metabolites within the organelles, sufficient for the western blot analyses of mTOR signaling which either co-exist with the organelle transiently or have fast components presented in Fig. 3 and 20 million cells are sufficient turnover rates. Using lysosomes as an example, we demonstrated the for lysosomal amino acids measurement shown in Fig. 4. In feasibility of monitoring changes in mTORC1 signaling in response contrast, most density gradient ultracentrifugation methods require to nutrient (amino acid) availability and of quantifying the contents typically 200–500 million cells and a time duration of several hours of individual amino acid species (Figs 3 and 4). Third, the (de Araujo and Huber, 2007; Michelsen and von Hagen, 2009). genetically encoded twin strep tag can also be used in animals to Furthermore, our method requires minimal equipment and experience, assess the physiological functions of specific organelles in vivo.It and can be performed in any biomedical laboratory. Finally, the twin has been shown that mitochondria from specific cell types can be strep purification is expected to cost less than antibody-based quickly isolated with the use of a mitochondrion-targeted epitope approaches, due to the high yield of organelle recovery shown in tag from complex tissues without cell sorting, which increases the our study (thus fewer beads are needed) and excellent stability of speed of isolation and allows better retention of the mitochondrial streptavidin (Bayer et al., 1990; Kimple et al., 2013). metabolite profile (Ahier et al., 2018; Bayraktar et al., 2019). We The method described in the current study can be of great use in expect that the tools described here can further increase the understanding the structures and functions of intracellular capability of in vivo profiling of different organelles in different organelles. First, it is known that there are quantitative and cells and tissues. Finally, the twin strep tag can be used in qualitative differences in protein levels in the same type of combination with other tags, such as 3×HA, for rapid isolation of organelles from different cell types (Itzhak et al., 2017). The different organelles in the same cells or tissues. simplified procedures and reduced requirement for cell numbers in While most amino acid species showed similar proportions among our approach make it possible to isolate and profile the composites the total amino acids in the lysosomal lumen and in the cytosol in our of proteins, lipids and small molecule metabolites in parallel and in lysosomal amino acid measurement, a few amino acids showed the same organellar population from different cell types. This kind preferential lysosomal accumulations. Our overall conclusion is of approach might be extremely important for cancer studies. There consistent with a previous lysosomal amino acid measurement that are overwhelmingly increasing collections of information on also showed differential distribution of some amino acids in the Journal of Cell Science

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Fig. 6. Characterization of isolated mitochondria and peroxisomes and determination of factors critical to the organelle purification. (A,B) Western blot analyses for the purity of mitochondrial (A) and peroxisomal (B) preparations using intracellular organelle markers as in Fig. 2C. (C,D) Effect of bead diameter on the 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 were determined 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 by comparing the densities of SDHA and catalase, respectively, in the purified organelles to that in PNS on the same Western blots. cytoplasm and lysosomal lumen (Abu-Remaileh et al., 2017). Semenkovich, 2014), respectively. It is possible that the isolation However, there are some obvious differences between the present efficiency can be further increased with optimal sized beads for each study and that of Abu-Remaileh et al. For example, the relative organelle type. For experiments in which the maximal recovery is proportions of cysteine, alanine, aspartate, arginine and valine are desired, we suggest performing a pilot experiment using bead sizes significantly higher in the lysosomal lumen than in cytosol in HeLa between 10 nm and 200 μm to identify the bead size that yields the cells in our study, whereas cystine is more abundant in lysosomal highest recovery efficiency. It may also be possible to predict the most lumen of HEK 293 cells (Abu-Remaileh et al., 2017). The different appropriate bead size for an organellar population by mathematical distributions of amino acids in the lysosomal lumen in these two cell modeling of maximal surface interactions between the desired types may be due to different expression levels of various lysosomal organelles and the beads. We performed most of our experiments amino acid transporters and different experimental conditions. using 1 μm magnetic streptavidin beads from Thermo Fisher However, the exact reasons remain to be further investigated. Scientific. It is reported that the twin strep tag has a picomolar Kd One interesting finding from our study is that the efficiency of towards Strep-Tactin XT, a mutant form of streptavidin (Yeliseev organelle isolation is dependent on the size of the magnetic beads. et al., 2017). Unfortunately, except for the 50 nm sized beads, Among the beads of different diameters, 50 nm, 1 μm, 5 μm, and Strep-Tactin XT beads smaller than 1 μmindiameterwerenot 25 μm, tested in the current study, we found that the 1 μm diameter commercially available at the time of our study. We anticipate that beads performed the best for the isolation of lysosomes, mitochondria both the yield and speed of purification will be further improved by and peroxisomes. This result suggests that comparable sizes between Strep-Tactin XT beads of more appropriate sizes. the beads and organelles are critical to the success of affinity In summary, we describe a rapid and efficient method for the purification of organelles, likely due to the maximal occupation (and/ isolation of intact lysosomes, mitochondria and peroxisomes. We or exclusion) of organelles on the beads. Lysosomes, mitochondria provide evidence that lysosomes purified by this method can be and perixosomes, have a diameter of 0.5–1 μm (Xu and Ren, 2015), used to analyze signaling dynamics and metabolites. Predictably,

0.75–3 μm (Wiemerslage and Lee, 2016) and 0.1–1 μm (Lodhi and this method should also be compatible for quick isolation of specific Journal of Cell Science

7 TOOLS AND RESOURCES Journal of Cell Science (2019) 132, jcs235390. doi:10.1242/jcs.235390 organellar populations from other mammalian tissues or non- Isolation of lysosomes, mitochondria and peroxisomes mammalian cells or tissues, such as those of yeast, plant, C. elegans Approximately 6×106 HeLa cells stably expressing Lyso-2Strep, Mito- or Drosophila, although organism-specific organelle targeting 2Strep or PEX-2Strep, were seeded on a 150-mm cell culture dish overnight μ sequence might be needed to target the twin strep fusion proteins and 1 g/ml of doxycycline was added at the time of seeding. On the day of to the desired organelles. experiment, medium was replaced with fresh medium free of doxycycline ∼3 h before the experiment. All organelle isolation procedures were performed in a cold room with ice-cold reagents. Cells were washed twice MATERIALS AND METHODS with PBS, collected in 1 ml of potassium phosphate-buffered saline (KPBS; General reagents and antibodies 136 mM KCl, 10 mM KH2PO4, pH 7.3) with a cell lifter, and then Streptavidin beads were purchased from Thermo Fisher Scientific transferred into a 1.5 ml centrifuge tube. The cells were centrifuged for (Waltham, MA, cat. #88817) and Creative Diagnostic (Shirley, NY; 1 min at 1000 g and then resuspended in 1 ml of KPBS. Resuspended cells 50 nm, cat. #WHM G066; 1 µm, cat. #WHM-S103; 5 µm, cat. #WHM- were homogenized in a 2 ml glass tissue grinder (VWR, Radnor, PA) with S108; 25 µm, cat. #WHM-S177), while Tactin beads were purchased from 30 gentle and continuous strokes. The homogenates were centrifuged at IBA biosciences (Gottingen, Germany, cat. #6-5510-050). Beads were 1000 g for 2 min and 800 μl of the post-nuclear supernatant (PNS) was washed with phosphate-buffered saline (PBS) before use. LysoTracker was added to 150 μl or 300 μl of prewashed streptavidin-conjugated magnetic purchased from Thermo Fisher Scientific (Waltham, MA). Antibodies for beads (cat. #88817, Thermo Fisher Scientific) in a 1.5 ml centrifuge tube. S6K (cat. #9202, 1:1000), SDHA (cat. #5839, 1:1000), catalase (cat. The PNS and beads were gently mixed and incubated in a tube rotator for #12980, 1:1000), PDI (cat. #3501, 1:1000), Golgin-97 (cat. #13192, 0.5–30 min to allow binding. After incubation, the beads were collected 1:1000) and ERK1/2 (cat. #4696, 1:1000) were from Cell Signaling with a magnetic stand (cat. #12321D, Thermo Fisher Scientific). The Technology (Danvers, MA); antibody for SERCA (cat. #SC-271669, supernatant was discarded and beads resuspended in 1 ml KPBS and then 1:1000) was from Santa Cruz Biotechnology (Dallas, TX); antibody for transferred to a new 1.5 ml tube. The beads were washed two more times by LAMP1 (cat. #L1418, 1:2500) was from Sigma-Aldrich (St Louis, MO); resuspending in 1 ml KPBS and pelleting with the magnetic stand. For antibodies for PMCA2 (cat. #19678-1-AP, 1:1000), ATP1A1 (cat. #14418-1- imaging, the organelles were eluted from the beads by incubation with AP, 1:1000), GOLM1 (15126-1-AP, 1:1000), SLC27A2 (14048-1-AP, 100 μl of 20 mM biotin in KPBS for 10 min, followed by collecting the 1:1000) and TOM20 (cat. #11802-1-AP, 1:10,000) were from ProteinTech beads with the magnetic stand and saving the supernatant. (Rosemont, IL); and antibody for LAMP2 (cat. #9840-01, 1:2500) was from the Developmental Studies Hybridoma Bank (Iowa City, IA). Dylight Western blotting 800-conjugated goat anti-mouse-IgG (cat. #SA5-10176, 1:5000) and Dylight Isolated organelles on the beads or PNS were mixed with an equal volume of 680-conjugated goat anti-rabbit-IgG (cat. #35568, 1:5000) were from Thermo 2×SDS sampling buffer. Protein samples were separated by SDS-PAGE and Fisher Scientific. All restriction enzymes and Q5® High-Fidelity DNA transferred onto nitrocellulose membranes. Membranes were blocked with Polymerase for PCR were from New England Biolabs (Ipswich, MA). 1% 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% Molecular cloning casein in TBS supplemented with 0.1% Tween-20 (TBST), followed by LAMP1, mGFP and the twin strep tag, were amplified by PCR from LAMP- fluorescently labeled secondary antibodies. The fluorescent signals were CFP-FKBP (Komatsu et al., 2010), mGFP-PASS (Lu et al., 2016; Zhang detected with the Li-COR Odyssey infrared imaging system from Li-COR et al., 2014), and AAVS1_Puro_PGK1_3xFLAG_Twin_Strep (Addgene Biotechnology (Lincoln, NE). The signal intensities of bands were analyzed #68375; Dalvai et al., 2015), respectively, and fused by PCR to generate the with Image Studio Lite (Li-COR). The median value of blank space around LAMP1–mGFP–2strep fusion protein. The Lyso-strep is then cloned into each band was set as the background and subtracted from the corresponding LT3G-mGFP-PASS (Lu et al., 2016) derived from L3GEPIR (Fellmann band intensities. Western blots are representatives of at least three biological et al., 2013) that contains the third generation tetracycline-inducible replicates from independent experiments with similar results. promoter, to generate LT3G–LAMP1–mGFP–2strep (Lyso-2strep). The cDNA encoding amino acids 1–34 of TOM20 was amplified by PCR from Fluorescence microscopy TOM20-CFP-FRB (Komatsu et al., 2010) and then cut with NheI and HeLa cells expressing the organelle probes were stained with antibodies BamHI at the artificially introduced sites. To generate LT3G–Mito–mGFP– specific for individual organelles, and visualized with a Nikon A1 confocal 2strep (Mito-2Strep), LT3G–LAMP1–mGFP–2strep was cut with NheI and microscope. Cells, purified lysosomes or mitochondria, were incubated with BamHI, and the LAMP1 fragment was replaced with the TOM20 fragment. 1 μM LysoTracker (Thermo Fisher Scientific), MitoTracker (Cell Signaling The cDNA encoding amino acids 1–42 of PEX3 was amplified by PCR Technology) or TMRE (Sigma-Aldrich) for 15 min at room temperature from pβactin-PEX3-mRFP (Kapitein et al., 2010) and then cut with NheI before imaging. For CCCP treatment, after the third wash during and BamHI at the artificially introduced sites. To generate LT3G–Pex– mitochondrial isolation, beads were resuspended in 500 µl KPBS mGFP–2strep (Pex-2Strep), the LAMP1 fragment of LT3G–LAMP1– containing 2 mM sodium pyruvate and 50 µM CCCP and incubated at mGFP–2strep was removed by NheI and BamHI digestion, and then room temperature for 30 min. TMRE was then added to the solution to a replaced with the cut PEX3 fragment. The full sequences and maps of these final concentration of 1 µM and incubated at room temperature for 15 min. constructs will be shared upon request. After incubation, beads were washed twice in 500 µl KPBS containing 2 mM sodium pyruvate and 50 µM CCCP. Mitochondria not treated with Cell culture, lentivirus production and transduction CCCP were processed in parallel for the same duration and temperature HeLa cells from ATCC (Manassas, VA) were maintained in Dulbecco’s exposure. The bound mitochondria were eluted from beads in 100 µl 20 mM modified Eagle’s medium (DMEM) plus 10% fetal bovine serum (FBS) biotin in KPBS containing 2 mM sodium pyruvate for 10 min at room (Thermo Fisher Scientific). Cells were passaged less than five passages temperature. Fluorescence images are representatives of at least three before being used for the current study. For amino acid starvation, cells were independent experiments with similar results. treated with amino acid-free DMEM (US Biological) supplemented with 10% FBS that has been dialyzed with Slide-A-Lyzer™ Dialysis Cassettes Sample preparation for mass spectrometry analysis with 3.5 kDa cut-off (Thermo Fisher Scientific). Lentiviruses were collected of amino acids from TLA-293T cells (Thermo Fisher Scientific) co-transfected with the After the final wash, beads bound with lysosomes were incubated with 60 µl lentiviral vector (Lyso-2Strep, Mito-2Strep, or Pex-2Strep), pCMV-dR8.2 of 50% methanol on ice for 5 min. Beads were then collected with a and pMD2.G using Lipofectamine and Plus reagent (Thermo Fisher magnetic stand and eluates were transferred to a clean glass vial (VWR). For Scientific) as described previously (He et al., 2017; Wang et al., 2017). At 2 cytosolic amino acids, the cytosol was prepared by centrifugation of PNS at days after infection with lentiviruses, HeLa cells were selected with 16,000 g for 3 min at 4°C to precipitate all organelles. Then 40 µl of the

puromycin (1 μg/ml) for stable expression. supernatant was mixed with 40 µl 100% liquid chromatography and mass Journal of Cell Science

8 TOOLS AND RESOURCES Journal of Cell Science (2019) 132, jcs235390. doi:10.1242/jcs.235390 spectrometry (LC-MS) grade methanol. The samples for LC-MS analysis Bayraktar, E. C., Baudrier, L., Özerdem, C., Lewis, C. A., Chan, S. H., Kunchok, were prepared by spiking 5 µl of isotopic labeled standard mix into each T., Abu-Remaileh, M., Cangelosi, A. L., Sabatini, D. M., Birsoy, K. et al. (2019). sample. MITO-tag mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo. Proc. Nat. Acad. Sci. USA 116, 303-312. doi:10. 1073/pnas.1816656115 Reagents and internal standards for mass spectrometry Bhola, P. D. and Letai, A. (2016). Mitochondria-judges and executioners of cell High-performance liquid chromatography (HPLC)-grade acetonitrile, death sentences. Mol. Cell 61, 695-704. doi:10.1016/j.molcel.2016.02.019 methanol and water were procured from Burdick & Jackson (Morristown, Braulke, T. and Bonifacino, J. S. (2009). 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Absolute quantification of matrix metabolites reveals the dynamics of mitochondrial 0.1% formic acid (buffer A) and 0.1% formic acid in acetonitrile (buffer B). metabolism. Cell 166, 1324-1337.e11. doi:10.1016/j.cell.2016.07.040 The samples were analyzed on 6490 triple quadrupole mass spectrometer Chen, W. W., Freinkman, E. and Sabatini, D. M. (2017). Rapid immunopurification coupled with 1290 series HPLC system equipped with a degasser, binary of mitochondria for metabolite profiling and absolute quantification of matrix pump, thermostatted auto sampler and column oven (Agilent Technologies). metabolites. Nat. Protoc. 12, 2215-2231. doi:10.1038/nprot.2017.104 Data analysis was carried out by using Agilent Mass Hunter workstation Dalvai, M., Loehr, J., Jacquet, K., Huard, C. C., Roques, C., Herst, P., Cote, J. software. All the identified amino acids were normalized to the levels of and Doyon, Y. (2015). 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R., Bergschneider, E., von Toerne, C., Hauck, S. M., The authors declare no competing or financial interests. Aichler, M., Walch, A. K., Wurst, W., Wiesner, R. J., Johnston, I. C. D. et al. (2013). Efficient isolation of pure and functional mitochondria from mouse tissues Author contributions using automated tissue disruption and enrichment with anti-TOM22 magnetic Conceptualization: G.D.; Methodology: J.X., J.H., W.P.X., E.H., C.S.R.A., N.P., beads. PLoS ONE 8, e82392. doi:10.1371/journal.pone.0082392 H.-E.K., M.X.Z., G.D.; Validation: W.P.X.; Formal analysis: J.X., J.H.; Investigation: Frezza, C., Cipolat, S. and Scorrano, L. (2007). Organelle isolation: functional J.X., J.H., G.D.; Resources: C.S.R.A., N.P., H.-E.K.; Data curation: J.X., M.X.Z., mitochondria from mouse liver, muscle and cultured fibroblasts. Nat. Protoc. 2, G.D.; Writing - original draft: J.X., G.D.; Writing - review & editing: J.X., M.X.Z., 287-295. doi:10.1038/nprot.2006.478 G.D.; Supervision: M.X.Z., G.D.; Project administration: G.D.; Funding acquisition: Graham, J. M. (2001a). Isolation of lysosomes from tissues and cells by differential N.P., M.X.Z., G.D. and density gradient centrifugation. Curr. Protoc. Cell Biol. Chapter 3, Unit 3.6. doi:10.1002/0471143030.cb0306s07 Funding Graham, J. M. (2001b). Isolation of peroxisomes from tissues and cells by This study was supported in part by grants from American Heart Association differential and density gradient centrifugation. Curr. Protoc. Cell Biol. Chapter 6, (19TPA34910051 to G.D.), National Institutes of Health (AR075830 to G.D., and Unit 3.5.1-3.5.22. doi:10.1002/0471143030.cb0305s06 NS092377 to M.X.Z. and P30 CA125123 to N.P.), Cancer Prevention and Research Graham, J. M. (2001c). 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