Flotillins bind to the dileucine sorting motif of b-site amyloid precursor protein-cleaving enzyme 1 and influence its endosomal sorting Bincy A. John, Melanie Meister, Antje Banning and Ritva Tikkanen Institute of Biochemistry, Medical Faculty, University of Giessen, Germany

Keywords Alzheimer’s disease; endocytic cargo; endocytosis; endosomal sorting; b-secretase Correspondence R. Tikkanen, Institute of Biochemistry, Medical Faculty, University of Giessen, Friedrichstrasse 24, 35392 Giessen, Germany Fax: +49 641 9947 429 Tel: +49 641 9947 420 E-mail: [email protected]. uni-giessen.de (Received 25 September 2013, revised 12 February 2014, accepted 19 February 2014) doi:10.1111/febs.12763

The b-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a protease that participates in the amyloidogenic cleavage of the Alzheimer amyloid precursor protein. Trafficking of BACE1 has been shown to be largely mediated by an acidic cluster dileucine motif in its cytoplasmic tail. This sorting signal functions both in endocytosis and endosomal sorting/recycling of BACE1 by providing a binding site for various sorting factors, such as the Golgi-localizing c-ear containing ADP ribosylation factor binding (GGA) proteins that mediate BACE1 sorting within endosomes. Because flotillin-1 has been suggested to bind to BACE1 cytoplasmic tail, we analyzed the role of flotillins in BACE1 sorting. We show that flotillin1 directly binds to the dileucine motif in the cytoplasmic tail of BACE1, whereas flotillin-2 binding is mainly mediated by its interaction with flotillin-1. Depletion of flotillins results in altered subcellular localization of BACE1 in endosomes and stabilization of BACE1 protein. Furthermore, amyloidogenic processing of Alzheimer amyloid precursor protein is increased. Flotillins compete with GGA proteins for binding to the dileucine motif in the BACE1 tail, suggesting that they play an important role in endosomal sorting of BACE1. The present study shows for the first time that flotillins are involved in endosomal sorting of BACE1. Because the endosomal localization of BACE1 affects its function as the b-secretase by increasing amyloidogenic processing of the amyloid precursor protein, flotillins may play a novel role in Alzheimer’s disease. The present study is the first to show that flotillins bind to a canonical sorting signal and influence the binding of endosomal sorting factors onto cargo tails. Structured digital abstract • BACE1 and flotillin-2 physically interact by proximity ligation assay (View interaction) • BACE1 and flotillin-1 physically interact by proximity ligation assay (View interaction) • BACE1 binds to flotillin-1 by pull down (View interaction) flotillin-2 physically interacts with BACE1 by anti bait coimmunoprecipitation (View interaction)

Abbreviations ACDL, acidic cluster dileucine; AD, Alzheimer’s disease; APP, Alzheimer amyloid precursor protein; Ab, amyloid b; BACE1, b-site amyloid precursor protein-cleaving enzyme 1; CD-MPR, cation dependent mannose 6-phosphate receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GGA, Golgi-localizing c-ear containing ADP ribosylation factor binding protein; GST, glutathione S-transferase; LAMP3, lysosome associated membrane protein 3; PLA, proximity ligation assay; shRNA, short hairpin RNA; siRNA, short interfering RNA; TGN, trans-Golgi network; WT, wild-type.

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Introduction Alzheimer’s disease (AD) is a common, age-associated neurodegenerative disease that manifests as progressive dementia and loss of mental skills. A major characteristic of AD is the accumulation of specific plaque structures in the brain. These extracellular ‘senile plaques’ contain the so-called amyloid b (Ab) peptide, which is a processing product of a transmembrane protein termed Alzheimer amyloid precursor protein (APP). The Ab peptide is generated by a sequential proteolytic processing of APP by two distinct proteases that are termed b- and c-secretase [1]. The c-secretase is a multi-subunit complex that cleaves APP within its transmembrane domain, whereas the b-secretase is a single-span transmembrane protein with a cleavage site within the ectodomain of APP [2]. The b-secretase, also called b-site amyloid precursor protein-cleaving enzyme 1 (BACE1) or memapsin 2, belongs to the family of aspartyl proteases [3–5]. BACE1 evidently cleaves APP in an acidic endosomal compartment after endocytosis [6], thereby facilitating Ab peptide generation. Sorting of transmembrane proteins is generally controlled by sorting signals in the cytoplasmic domains of the cargo proteins. Various types of sorting signals exist, among them the acidic cluster dileucine (ACDL) type of signals and tyrosine-based motifs that can mediate both endocytosis and endosomal sorting [7]. Various types of adaptor proteins, including the tetrameric adaptin complexes and proteins of the Golgilocalizing c-ear containing ADP ribosylation factor binding protein (GGA) family, mediate cargo sorting by means of binding to the sorting signals and facilitating the recruitment of cargo into specific transport vesicles [8]. The cellular trafficking of BACE1 has been studied intensively. The short cytoplasmic tail of BACE1 with 23 amino acids contains a sorting signal of the ACDL type [9,10] (Fig. 1A). The two Leu residues in this determinant are important for the clathrin-mediated endocytosis of BACE1, whereas the acidic residues together with the Leu are required for the endosomal sorting and recycling of BACE1 back to the plasma membrane [9,10]. In addition, an adjacent Ser residue has been shown to be phosphorylated, which increases the recycling of BACE1 [11]. The ACDL motif binds to members of the GGA family (GGA1–GGA3) that are involved in the sorting of BACE1 [12]. Flotillin-1 and flotillin-2 are highly conserved proteins that have been suggested to function (e.g. in membrane receptor signaling, cell adhesion, cell migration and membrane trafficking processes) [13,14]. Flotillins FEBS Journal 281 (2014) 2074–2087 ª 2014 FEBS

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Fig. 1. Interaction of flotillins with the BACE1 cytoplasmic tail is dependent on the dileucine motif in vitro. (A) Human BACE1 cytoplasmic tail: letters in bold indicate the di-Leu sorting motif; letters in italics indicate the GGA binding motif. (B–D) Purified GST fusion proteins of the BACE1 (B) or CD-MPR (C) cytoplasmic tail (WT or LLAA) coupled to glutathione sepharose beads were incubated with lysates from HeLa cells in which flotillin expression was ablated by means of siRNAs. (D) Binding of purified recombinant flotillin-1 and flotillin-2 to BACE1 cytoplasmic tail (WT or LLAA) was tested. After SDS/PAGE, the respective proteins were detected by means of western blotting using specific antibodies. Ponceau staining was used to visualize the fusion proteins employed in the assay.

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are peripheral membrane proteins bound to the inner leaflet by means of myristoylation (only flotillin-2) and palmitoylation (both flotillins) [15,16], which mediate their association with specific cholesterol-rich microdomains in cell membranes. Their cellular localization is dynamic and flotillins are taken up from the plasma membrane and transported into endosomes upon growth factor stimulation [17,18]. Flotillins are mainly found in the cells in the form of flotillin-1/flotillin-2 hetero-oligomers that result from C-terminal interactions, mediated by the so-called flotillin domain [16,19]. Oligomerization appears to be important for the expression and stability of flotillin-1 because, in most cell types and in the knockout mouse models, depletion of flotillin-2 also results in a severe reduction of flotillin-1 levels. By contrast, the expression of flotillin-2 is less dependent on flotillin-1 [17,19–22]. Recent studies have revealed an apparent role for flotillins both in the regulation of signal transduction and in vesicular trafficking. We have shown that not only is flotillin-1 a novel scaffolder protein for the mitogen-activated protein kinases, but also it is required for clustering of the epidermal growth factor receptor at the plasma membrane upon signaling [23]. Furthermore, a role in endocytosis of various cargo proteins has been suggested for flotillins [24–28]. Flotillins have been implicated to play a role in AD and flotillin-1 accumulates in the brain of AD patients [29–33]. In line with this, depletion of flotillin-2 impairs the plasma membrane clustering of APP, thus negatively affecting the BACE1-mediated processing of an overexpressed APP variant carrying the so-called Swedish mutation, which is found in Alzheimer patients and normally exhibits increased amyloidogenic processing [27,34]. Although flotillin-1 was shown to bind to the cytoplasmic tail of APP [29], its depletion did not result in an impairment of APP endocytosis, different from the knockdown of flotillin-2 in the above-mentioned overexpression system [27]. These data are consistent with the recent findings obtained in flotillin double knockout mice carrying the same APP mutation together with a mutant presenilin-1, which only showed modest effects on APP processing upon the loss of both flotillins [35]. Interestingly, flotillin-1 coimmunoprecipitates with ectopically expressed BACE1 and its overexpression enhances the recruitment of BACE1 in membrane microdomains but inhibits its secretase activity [36]. However, it is not known whether this interaction is direct or mediated by other proteins. Furthermore, the role of flotillin-2 in the binding of BACE1 has not been studied, and there are no data available on the possible influence of flotillins with respect to the 2076

subcellular localization of BACE1. In the present study, we show that flotillin-1 directly binds to the dileucine motif in the cytoplasmic tail of BACE1, whereas flotillin-2 only shows an association mediated by flotillin-1. Flotillins compete with GGA2 for binding to the BACE1 tail and thus influence the endosomal sorting of BACE1. Importantly, the depletion of flotillins results in an altered endosomal localization of the wild-type (WT) BACE1, whereas the plasma membrane resident Leu to Ala (LLAA) mutant is not affected. BACE1 expression in HeLa cells is increased in cells stably depleted of flotillins, suggesting reduced degradation and an increased half-life of BACE1. Furthermore, amyloidogenic processing of APP is increased upon flotillin depletion, most likely as a result of increased BACE1. Thus, flotillins appear to be important for the cellular targeting of BACE1, which influences the amyloidogenic processing of APP. The results obtained in the present study provide important novel insights into the putative role of flotillins in AD as regulators of the membrane trafficking of BACE1.

Results Direct interaction of flotillins with BACE1 is mediated by the acidic cluster, dileucine sorting signal in the cytoplasmic tail of BACE1 A previous study by Hattori et al. [36] suggested that flotillin-1 associates with BACE1, and that flotillin-1 overexpression increases the association of BACE1 with detergent insoluble microdomains [36]. However, because the major parts of the cellular flotillin-1 and flotillin-2 are associated as hetero-oligomers in most cells [17,25] and the study of Hattori et al. [36] did not address the role of flotillin-2 in BACE1 binding, we carried out pulldown experiments using purified glutathione S-transferase (GST) fusions of the cytoplasmic domain of human BACE1. The short cytoplasmic tail (23 amino acids) of BACE1 was fused to the C-terminus of GST (Fig. 1A). In addition to the WT tail, we also tested the interaction of flotillins with the mutated tail in which the dileucine within the ACDL was mutated into alanines (LLAA; Fig. 1A shows the mutated Leu in bold). To study the involvement of each flotillin in the binding, HeLa cells depleted of flotillins by means of short interfering RNA (siRNAs) were used for the pulldown (Fig. 1B). Flotillin-1 depleted cells express almost unchanged amounts of flotillin-2, whereas siRNA-mediated, acute flotillin-2 knockdown results in a profound depletion of flotillin1 as well, as a result of destabilization of the protein FEBS Journal 281 (2014) 2074–2087 ª 2014 FEBS

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[17,19]. In control cells, both flotillins were detected in the pulldown fraction of the WT tail, whereas the binding of flotillins to the LLAA mutant was clearly reduced. Interestingly, in cells depleted of flotillin-1, the binding of flotillin-2 to the WT tail of BACE1 was reduced to the same level as that observed with the LLAA tail in these and control cells. This implicates that flotillin-1 binds to the ACDL dileucine motif, whereas the binding of flotillin-2 is mainly mediated by flotillin-1. To test whether flotillins generally bind to acidic dileucine motifs, a pulldown with a GST fusion of the cytoplasmic tail of the cation dependent mannose 6phosphate receptor (CD-MPR) was used. Neither flotillin-1, nor flotillin-2 bound to the CD-MPR tail, whereas GGA2, which has been shown to interact with CD-MPR [37], was pulled down. This interaction was abrogated upon mutation of the LL motif to AA (Fig. 1C). To dissect the role of flotillins in BACE1 binding in more detail and to test whether the interaction is a direct one, we performed direct pulldown assays using bacterially expressed, purified proteins (Fig. 1D). Purified flotillin-1 again strongly bound to the WT BACE1-GST tail, whereas no binding was observed with the LLAA tail. Only a very weak binding that was close to the GST background level was observed with purified flotillin-2. Taken together, these data show that flotillin-1 directly interacts with the cytoplasmic tail of BACE1 and also that this interaction is mediated by the well known dileucine sorting motif in BACE1. To corroborate these findings in an in vivo context, we performed coimmunoprecipitation experiments with endogenous flotillins and BACE1 (Fig. 2A). For this, we used polyclonal flotillin-2 antibodies because they result in a quantitative precipitation of the pool of flotillin-1/flotillin-2 complexes in the cells. BACE1 was found to be immunoprecipitated with endogenous flotillin-2, verifying the relevance of the pulldown data. Because the signals observed in the coprecipitation experiment were very weak, we used a proximity ligation assay (PLA) to verify the interaction of endogenous BACE1 and flotillins. PLA is based on the use of specific antibodies and can detect two proteins that are at very close vicinity (< 40 nm) in cells and is generally used to study the interaction of proteins [38–40]. Interacting proteins are visualized as fluorescent signals whose localization in the cells can also be assessed, giving information on the compartment in which the interaction takes place. However, the signal intensities as such do not correlate with the strength or quantity of the interaction. Quantification of the signals is carried out by counting the FEBS Journal 281 (2014) 2074–2087 ª 2014 FEBS

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Fig. 2. Flotillins interact with endogenous BACE1. (A) Endogenous flotillin-2 was immunoprecipitated from HeLa cells and the coprecipitation of endogenous BACE1 was detected by western blotting. Caveolin-1 was used to control the specificity of the coprecipitation. (B) PLA with specific antibodies was used to detect the interaction of endogenous flotillin-1, flotillin-2 and BACE1 in HeLa cells. Red signals indicate the site of interaction; blue shows the nuclei. Panels show an overlay with phase contrast images. Upper row: left, flotillin-1 + BACE1; right, flotillin2 + BACE1 antibodies. Lower row, negative controls: left, staining performed with flotillin-2 antibody but without BACE1 antibody; right, staining of flotillin-2 knockdown cells with flotillin-2 and BACE1 antibodies. Scale bar = 10 lm.

amount of fluorescent signals per cells and comparing these with negative controls (performed without the first antibody). PLA analysis of endogenous flotillin-1 or flotillin-2 and BACE1 showed that the fluorescent dots that arise upon interaction of these proteins were mainly detected in the perinuclear region of the cells, with few dots residing in the cell periphery (Fig. 2B). Quantitative assessment of the data showed that 3.83
0.92 dots/cell were observed when flotillin-2 and BACE1 antibodies were used, whereas flotillin-1 and BACE1 labeling gave 3.78
0.39 dots. Three different negative controls were performed. When the antibody for BACE1 was omitted, we observed 0.104
0.030 dots, whereas omitting flotillin antibody resulted in 2077

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0.126
0.075 dots. Labeling of stably flotillin-2 depleted HeLa cells with flotillin-2 and BACE1 antibodies gave a labeling efficiency of 0.075
0.016 dots/cell. Thus, the negative controls showed, on average, almost 40-fold less dots than the samples labeled with two primary antibodies, and the difference between the samples and the negative controls was statistically significant in all cases (P < 0.001). Taken together, the results of the coimmunoprecipitation experiments and PLA show that endogenous flotillins and BACE1 interact, although this interaction appears not to be very prominent.

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Flotillin knockdown results in endosomal accumulation of WT but not LLAA mutant BACE1 Because the ACDL motif is important for both endocytosis and recycling of BACE1, we next tested whether a depletion of the cellular flotillin pool might affect the localization of BACE1. For this, we used HeLa cells in which flotillin expression was stably knocked down by means of lentivirus-mediated shRNA [17]. Flotillin-1 and flotillin-2 knockdown cells were transfected with WT and LLAA BACE1-myc, the localization of which was studied by immunostaining (Fig. 3A). The same settings were used during the imaging of all cells to allow for quantitative assessment of the stainings. In control shRNA cells, WT BACE1 was localized at the plasma membrane to a large degree. In addition, some perinuclear staining was seen, as described previously [41]. However, we did not observe any evident colocalization of flotillin-1 with WT BACE1. In flotillin knockdown cells, a more intense staining of the perinuclear region was observed, suggesting that flotillin depletion influences the cellular localization of BACE1. Quantification of BACE1 localization in control cells versus flotillin knockdown cells showed that, in a significantly higher fraction of cells, BACE1 was localized in the perinuclear region in flotillin knockdown cells (Fig. 3B). Consistent with the fact that the LLAA mutant does not bind to flotillins, plasma membrane localization was observed both in control and flotillin knockdown cells (Fig. S1). To dissect the localization of BACE1 in the absence of flotillins in more detail, we performed colocalization studies by means of immunofluorescent staining in HeLa cells stably depleted of flotillins (Fig. 4). Ectopically expressed BACE1-myc showed a prominent colocalization with the late endosome/multivesicular body marker lysosome associated membrane protein 3 (LAMP3/CD63) in flotillin knockdown cells, whereas much less colocalization was observed in the control cells (Fig. 4A; for the second pair of flotillin knockdown cells, see Fig. S2). Quantification of the fluorescence data showed that the Pearson coefficient for the 2078

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Fig. 3. Flotillin depletion influences the subcellular localization of BACE1-myc. Full-length, myc-tagged BACE1 was expressed in control and stable flotillin knockdown HeLa cells. (A) Cells were fixed in methanol and immunofluorescently labeled for myc-tag (red) and either flotillin-1 or -2 (green). Scale bar = 10 lm. (B) Quantification of the cells showing perinuclear staining for BACE1. For details, see Materials and methods. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001.

colocalization of LAMP3 with BACE1-myc was significantly higher in flotillin knockdown than in control cells (Fig. 4B). By contrast, BACE1 did not colocalize with the early endosomal marker Rab5 in control or FEBS Journal 281 (2014) 2074–2087 ª 2014 FEBS

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the endogenous protein, we immunostained BACE1 in human neuroblastoma SH-SY5Y cells stably depleted of flotillins. Consistent with the above data, BACE1 showed an increased perinuclear localization especially in flotillin-1 knockdown cells (Fig. 5A). In addition, the intensity of the staining appeared brighter, suggesting increased expression. Quantification of the fluorescence data using maximum intensity confocal sections showed that this was indeed the case, especially with the flotillin-1 knockdown cells, which demonstrated more than two-fold upregulation of BACE1 fluorescence (Fig. 5B). Flotillin-2 knockdown also showed a tendency towards higher expression, which, however, did not reach significance (Fig. 5B). To biochemically test whether flotillin depletion affects the expression of BACE1, endogenous BACE1 was detected by means of western blotting in HeLa cells stably depleted of flotillins. Note that, upon stable knockdown of flotillin-2, a considerable amount of flotillin-1 is still expressed in the stably depleted cells, in contrast to the siRNA-mediated depletion (Fig. 1A). Indeed, BACE1 expression was significantly (2.5- to 3.5-fold) increased in all four flotillin knockdown cell

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Fig. 4. Ectopically expressed BACE1 shows an enhanced localization to late endosomes in flotillin knockdown cells. (A) Control and stable flotillin knockdown HeLa cells that express fulllength, myc-tagged BACE1 were fixed and immunostained with anti-myc serum (green) and the late endosomal marker LAMP3/ CD63 (red). Scale bar = 10 lm. (B) Quantification of the colocalization of BACE1 with LAMP3. For details, see Materials and methods. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001.

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flotillin knockdown cells, nor was there any considerable colocalization with the Golgi marker Golgin-97 (Fig. S3). Flotillin-1 influences the localization and expression of endogenous BACE1 The above results suggested that flotillins are important for the correct cellular localization of overexpressed BACE1. To determine whether this is also true for FEBS Journal 281 (2014) 2074–2087 ª 2014 FEBS

Fig. 5. Cellular localization and stability of endogenous BACE1 is altered upon flotillin-1 knockdown. (A) Endogenous BACE1 was immunostained in SH-SY5Y cells in which flotillin expression was stably knocked down with lentiviral shRNAs. Scale bar = 10 lm. (B) Quantification of the staining for BACE1. For details, see Materials and methods. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001.

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lines (Fig. 6), thus verifying the impression of increased BACE1 staining in the absence of flotillins.

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Flotillins compete with GGA2 for the binding on BACE1 ACDL motif The dileucine motif in the BACE1 tail is also a binding site for the GGA proteins that have been shown to influence the endosomal trafficking of BACE1 [11,12,41,42]. Because flotillins and GGA proteins apparently share this binding motif, we tested whether flotillins and GGA2 cooperatively bind to BACE1 or whether they compete for the binding. For this, BACE1-GST pulldowns were again utilized. In line with previous findings [12,41], WT BACE1 tail bound GGA2, whereas the binding was abrogated by the LLAA mutation (Fig. 7A). Intriguingly, GGA2

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Fig. 6. BACE1 expression is increased in flotillin knockdown cells. (A) HeLa cells in which flotillin expression was stably knocked down were lysed and endogenous BACE1 was detected by means of western blotting. Flotillin depletion was associated with an increased expression of BACE1. (B) For quantification, BACE1 expression was normalized to GAPDH. Bar graphs show the mean
SD of six independent experiments. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001.

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Fig. 7. Flotillins compete for binding to the BACE1 cytoplasmic tail with GGA2, and flotillin depletion influences APP processing. (A) Purified GST fusion proteins of the BACE1 cytoplasmic tail (WT or LLAA) coupled to glutathione sepharose beads were incubated with lysates from stable flotillin knockdown HeLa cells. The binding of GGA2 and flotillins was tested using specific antibodies after SDS/PAGE and western blotting. Ponceau staining was used to visualize the fusion proteins employed in the assay. (B) Processing of APP in flotillin knockdown cells was measured using antibodies that recognize the C-terminal processing fragments of APP. (C) Quantification of the C-terminal APP processing fragments. Normalization was performed against GAPDH. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001.

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binding to WT BACE1 tail was increased in the absence of flotillins, suggesting that GGA2 and flotillins compete for binding to the dileucine motif in BACE1. Flotillin depletion results in increased processing of APP To test whether increased BACE1 expression also shows an effect on the processing of APP, we measured the C-terminal processing fragments of endogenous APP in the stable flotillin knockdown cells using an antibody that detects the C-terminus of APP. The amount of full-length APP was not significantly changed in these cells, whereas the C-terminal BACE1 processing fragment was significantly increased upon flotillin-2 knockdown (Fig. 7B,C). The C-terminal fragment was identified as the b-secretase fragment, C99, as a result of its size and reactivity with the 6E10 antibody, which recognizes the first 16 amino acids of the Ab peptide. Flotillin-1 knockdown cells also showed a tendency to higher APP processing (Fig. 7C). However, these data did not reach significance as a result of variation of the C99 amount between the different experiments. Nevertheless, these data show that flotillin knockdown increases the expression and endosomal localization of BACE1, which in turn results in increased amyloidogenic processing of APP.

Discussion The data obtained in the present study show that flotillins are directly involved in the cellular trafficking of BACE1. Hattori et al. [36] have previously demonstrated that flotillin-1 could be coprecipitated with overexpressed BACE1 from HEK293 cells, and flotillin-1 overexpression resulted in increased association of BACE1 with detergent-resistant membrane domains [36]. Unfortunately, the said study did not address the role of flotillin-2, nor did it show whether the binding of flotillin-1 to BACE1 is direct or indirect. Importantly, we provide evidence suggesting that endogenous flotillins and BACE1 interact. Our binding studies revealed a direct association of flotillin-1 with the dileucine motif in the BACE1 cytoplasmic tail, whereas the binding of flotillin-2 appears to be indirect. Because a large fraction of flotillins is associated with each other as hetero-oligomers, it is very likely that flotillin-2 binding to BACE1 is mediated by flotillin-1 to a large extent. However, a minor fraction of flotillin-2 was still able to bind to BACE1 tail in GST pulldown experiments with cell lysates depleted of flotillin-1, indicating that some of the flotillin-2 binding FEBS Journal 281 (2014) 2074–2087 ª 2014 FEBS

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may be independent of both flotillin-1 and the dileucine motif (see below). Depletion of either of the flotillins altered the subcellular localization of overexpressed and endogenous BACE1, resulting in perinuclear accumulation of BACE1 in structures that contained LAMP3 but not Rab5. These data suggest a novel role for flotillins in the intracellular/endosomal trafficking of transmembrane proteins. So far, mainly flotillin-1 has been postulated to be involved in either clustering at the plasma membrane or endocytosis of cargo proteins [23,24,27,28], although recent findings have also suggested that flotillins may be involved in recycling of E-cadherin and transferrin receptor in A431 cells [43]. However, little evidence for a direct role of flotillins in endosomal trafficking of receptor proteins has been presented. On the other hand, flotillin-1 has been suggested to be important in the intracellular sorting of lipids and thus also of lipid associated bacterial toxins such as cholera toxin, which binds to the ganglioside GM1 [44]. In line with this, intoxication of zebra fish with cholera toxin was shown to be flotillin dependent [45]. Another study implicated flotillins in the retrograde trafficking but not endocytosis of Shiga toxin and ricin and showed that flotillin depletion potentiates the effect of the toxins [46]. All three toxins are transported together with their lipid receptors from the plasma membrane to endosomes from which they are diverted towards the Golgi/ trans-Golgi network (TGN) and the endoplasmic reticulum. Interestingly, the retrograde trafficking of BACE1 from endosomes back to the plasma membrane has also been suggested to involve a detour through the Golgi/TGN [11,41], although the exact mechanism is not very well characterized. To our knowledge, this is the first time that flotillins have been implicated in endosome-Golgi transport of a transmembrane protein because all previous flotillin-dependent cargo molecules traffic by means of lipids. It is not known whether specific lipids are required for BACE1 transport to Golgi, although this may involve specific lipid microdomains that are characterized by flotillins. The ACDL motif in the BACE1 tail was also shown to be important for the binding of GGA proteins that regulate BACE1 trafficking [11,12,41,47]. GGAs also bind to the tail of other proteins containing similar ACDL motifs, such as CD-MPR [37]. However, flotillins specifically interacted with the ACDL motif of BACE1 but not of CD-MPR, suggesting that this interaction is cargo specific. Although the dileucine motif in BACE1 is also an endocytosis signal, uptake of WT BACE1 from the plasma membrane was not impaired upon depletion of either GGA protein [41] or 2081

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flotillins (present study), suggesting that neither GGAs, nor flotillins play a role in BACE1 endocytosis. However, the depletion of any of the three GGA proteins produces a BACE1 trafficking phenotype that phenocopies that of flotillin depletion, in that BACE1 accumulates in intracellular structures and is stabilized. We observed an increase in the BACE1 staining in flotillin knockdown cells, suggesting that the half-life of the protein may be increased. This was also supported by our western blotting findings showing that BACE1 amount was increased in cells stably depleted of flotillins. GGA3 plays a specific role in the transport of BACE1 towards lysosomes for degradation [47], whereas GGA1 and GGA2 are more likely to mediate endosome-TGN trafficking [12,41]. Interestingly, depletion of GGA3 has been shown to result in a similar stabilization and increase in BACE1 levels as a result of impaired lysosomal degradation [42,47], as observed in the present study upon flotillin depletion. However, in contrast to flotillin knockdown, which resulted in late endosomal/multivesicular body accumulation of BACE1, GGA3 depletion enhanced the early endosomal localization of BACE1 [42]. Furthermore, GGA3dependent regulation of BACE1 levels is independent of the ACDL motif [42]. Thus, if the lysosomal targeting of BACE1 was specifically impaired upon flotillin knockdown, increased early endosomal localization should be observed. However, because BACE1 accumulated in late endosomes/multivesicular bodies upon flotillin ablation and flotillin-1 competed with GGA2 for binding to the BACE1 ACDL, it is likely that flotillins are also required for the sorting of BACE1 into the recycling pathway. Both GGA proteins and flotillin-1 bind to the same sorting signal in BACE1 (Fig. 1A). This raises the question of whether the binding of GGAs and flotillin1 is cooperative or competitive. Interestingly, increased binding of GGA2 to BACE1 ACDL was observed in the absence of flotillins, suggesting that GGA2 and flotillin-1 compete for the same binding site. We do not know in which order GGAs versus flotillins bind to BACE1 to facilitate its endosomal sorting. However, because depletion of either flotillins or GGA proteins results in endosomal accumulation of BACE1, and flotillin depletion does not prevent but enhances GGA2 binding, it is more likely that GGA2 binds to BACE1 first and is then released upon binding of flotillin-1 to the ACDL. In this scenario, the binding of flotillin-1 to BACE1 is a prerequisite for the sorting, and it might be required, for example, for the recruitment of BACE1 in endosomal microdomains formed by flotillins. This hypothesis is supported by the data obtained in the study by Hattori et al. [36] who showed an 2082

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increased association of BACE1 with membrane microdomains upon flotillin-1 overexpression [36]. Further studies will be required to clarify the exact binding mode and order of flotillin-1 and GGAs to BACE1, as well as the effect of flotillins on BACE1 recruitment into endosomal microdomains. Previous studies have shown that the depletion of especially flotillin-2 results in altered trafficking and processing of APP [27], in line with the findings suggesting that flotillins bind to the cytoplasmic tail of APP [unpublished data, BAJ; 29]. Although a direct effect on APP trafficking by flotillins is plausible in the light of these data, the results of the present study suggest that flotillins also influence APP processing indirectly, by means of altering the subcellular trafficking and the amount of BACE1. In the present study, we observed an increased amyloidogenic processing of APP in flotillin knockdown cells, which is probably a direct consequence of the increased amount of BACE1 in these cells. Our data are also well in line with those of Tesco et al. [47] who observed an increase in the APP C99 fragment upon an increased amount of BACE1 [47]. Flotillin-2 depletion in neuronal cells has been suggested to result in decreased amyloidogenic processing of APP, which was explained by the plasma membrane retention of APP [27]. However, these data are not directly comparable with those of the present study because the processing of APP was measured using an overexpressed, mutant APP (so-called Swedish mutant) [34] whose processing and trafficking is profoundly different from the endogenous, WT protein. In addition, in the different cell types used (neuronal versus epithelial), APP processing might be considerably distinct. Thus, further studies using the recently generated knockout mouse models [20,22,35] are required to clarify the potential in vivo effects of flotillin ablation on APP processing. In the present study, we show that flotillins bind to the dileucine sorting motif of BACE1 and that flotillin depletion affects the cellular localization and expression level of BACE1. Furthermore, flotillin binding in the BACE1 tail modulates the binding of GGA proteins, which is important for the correct cellular sorting of BACE1 because flotillin-1 competes for binding to the BACE1 ACDL with GGA2. These findings not only have important implications for AD because impaired cellular trafficking of BACE1 may result in an increased amyloid load, but also show for the first time that flotillins bind to a canonical sorting signal of a cargo protein and thereby influence the binding of endosomal sorting factors onto cargo tails. Thus, our findings also suggest a novel role for flotillins in the sorting of cargo within endosomal structures. FEBS Journal 281 (2014) 2074–2087 ª 2014 FEBS

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Materials and methods Antibodies and DNA constructs Mouse monoclonal anti-flotillin-1, anti-flotillin-2, anti-caveolin-1 and anti-GGA2 sera were obtained from BD Transduction Labs (Heidelberg, Germany); rabbit polyclonal anti-c-myc and Rab5 sera, as well as mouse monoclonal anti-LAMP3/CD63 serum, was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); mouse monoclonal anti-GAPDH serum was obtained from Abcam (Cambridge, MA, USA); and mouse monoclonal anti-c-myc serum was obtained from Cell Signaling (Danvers, MA, USA). Mouse monoclonal antibody against Golgin-97 was obtained from Life Technologies (Darmstadt, Germany). A polyclonal rabbit antibody against the cytoplasmic domain of APP (Calbiochem, Darmstadt, Germany) and the 6E10 monoclonal antibody against the first 16 amino acids of the Ab peptide (Covance, M€ unster, De) were used to detect full-length APP (110 kDa) and APP-C99 (12 kDa). The BACE1 antibody was kindly provided by W. Annaert (VIB Center for Biology of Disease, Leuven, Belgium). The primary antibodies used for immunofluorescence were detected with Cy3-conjugated goat anti-mouse serum (Jackson Immunoresearch, Newmarket, UK) and with Alexa Fluor 488 donkey anti-rabbit serum (Life Technologies). Human full-length BACE1-myc (WT and LLAA, GenBank: NM_012104) expression constructs were kindly provided by G. Tesco (Tufts University School of Medicine, Boston, MA, USA). The cytoplasmic domain of BACE-1 was PCR amplified from these constructs and cloned into pGEX-4T1 (GE Healthcare, Munich, Germany) for the expression of GST fusions of the BACE1 tail. The cytoplasmic tail of the CD-MPR (GenBank: NM_002355.3) was cloned with a similar strategy in pGEX-4T1. Rat flotillin-1-GST (GenBank: U60976) and flotillin-2-GST (GenBank: AF023302) coding regions were cloned into pET41a expression vector (Novagen, Darmstadt, Germany).

Cell culture and generation of stable knockdown cells Human cervix adenocarcinoma cells (HeLa) were maintained in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Life Technologies), 100 unitsmL–1 penicillin and 100 lgmL–1 streptomycin at 8% CO2 and 37 °C. Human neuroblastoma SH-SY5Y cells were cultured in RPMI medium with 10% fetal bovine serum and antibiotics as above at 5% CO2. Stable knockdown of flotillins in SH-SY5Y and HeLa cells was performed as described previously [17] using the Mission Lentivirus RNAi system (Sigma-Aldrich, Taufkirchen, Germany). After selection, the pool of survived cells was used to avoid clonal effects. Knockdown was verified by means of western blotting and immunostaining. Flotillin-1

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and -2 expression was downregulated using siRNAs (StealthTM siRNA system; Life Technologies) as described previously [23]. As a control, an oligo that does not target any human sequence (StealthTM RNAi Negative Control, medium GC content) was used.

Cell lysis The cells were lysed in cell lysis buffer (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 or Nonidet-P40, 60 mM N-octyl-b-D-glucopyranoside) supplemented with protease inhibitors (Sigma-Aldrich). Protein concentrations were determined, and equal protein amounts were analyzed by 10% SDS/PAGE. Western blotting was performed with specific primary and secondary horseradish peroxidase coupled antibodies.

Purification of recombinant proteins Bacterial expression vectors encoding for GST, BACE1CT-GST, BACE1-CT-LLAA-GST, MPR46-CT-GST, MPR46-CT-LLAA-GST, flotillin-1-GST and flotillin-2GST were transformed into a bacterial expression strain. Overexpression of flotillin-1- and flotillin-2-GST was induced by adding 0.25 mM isopropyl thio-b-D-galactoside into a liquid culture with D600 of 0.4 overnight at 19 °C, whereas other GST fusion proteins were induced with 1 mM isopropyl thio-b-D-galactoside for 6 h at 37 °C. The bacteria were harvested by centrifugation and lysed in GST lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 5% glycerol, 0.1% Nonidet P-40) supplemented with 100 lgmL–1 lysozyme, 1 mM phenylmethanesulfonyl fluoride, 1 mM dithiothreitol and 1 mM protease inhibitors (aprotinin, leupeptin, pepstatin). The GST proteins were bound to glutathione sepharose beads (GE Healthcare).

GST pulldown with cell lysates GST fusion proteins were immobilized on glutathione Sepharose 4B beads (GE Healthcare). HeLa cells were lysed in cell lysis buffer supplemented with protease inhibitors (Sigma-Aldrich) and 60 mM N-octyl-b-D-glucopyranoside. The cell lysate was centrifugated and then incubated with the immobilized GST fusion proteins overnight at 4 °C with rotation. The beads were washed three times with 1 mL of lysis buffer. Proteins bound to the beads were eluted by boiling in SDS sample buffer containing 50 mM dithiothreitol for 5 min and separated by 10% SDS/PAGE .

Direct GST pulldown For direct GST pulldown experiments, BACE1-GST (WT and LLAA) was coupled to GSH Sepharose. Flotillin1-GST or flotillin-2-GST was cleaved with 2 units of

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thrombin to remove the GST tag. Cleavage was performed with 20 lg of purified GST fusions bound to Sepharose beads in 20 mM Tris-HCl (pH 8.4), 0.15 mM NaCl, 2.5 mM CaCl2 freshly supplemented with 1 mM dithiothreitol. The reaction was carried out overnight at 19 °C. Thrombin was inactivated with 1 mM phenylmethyl sulfonylfluoride, and the GST bound beads were removed by centrifugation. The supernatants containing untagged flotillin-1 or flotillin-2 were mixed with 5 lg of the GST fusion of BACE1 or GST and rotated at 4 °C for 3 h. After washing (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.01% Triton X-100), the beads were boiled in SDS sample buffer for 5 min and separated by SDS/ PAGE.

Coimmunoprecipitation HeLa cells were lysed in cell lysis buffer supplemented with protease inhibitors. Unspecifically, binding material was removed with 50 lL of Pansorbin beads (Calbiochem). The supernatants were incubated overnight at 4 °C with 30 lL of Dynabeads Protein A (Life Technologies) coupled with polyclonal rabbit anti-flotillin-2 serum. The beads were washed three times in 1 mL of lysis buffer, mixed with SDS sample buffer and denatured at 94 °C for 5 min. The precipitated fraction was analyzed by SDS/PAGE. Endogenous BACE1 was detected by western blotting with mouse monoclonal antibody.

PLA Immunocytochemistry and quantification Control and stable flotillin knockdown HeLa cells grown on coverslips were transiently transfected with 500 ng of BACE1-myc (WT or LLAA) or control vector per coverslip, fixed in methanol for 10 min at 20 °C, washed with NaCl/Pi and blocked with 1% BSA in NaCl/Pi (blocking buffer). The cells were then incubated with primary antibodies in blocking buffer for 1 h and subsequently washed with NaCl/Pi, and the primary antibodies were detected with various fluorophore (Cy3 or Alexa 488) coupled secondary antibodies. The nuclei were counterstained with 40 ,6-diamidino-2-phenylindole. The specimens were then embedded in Gelmount (Biomeda, Foster City, CA, USA) supplemented with 50 mgmL1 1,4-diazadicyclo(2,2,2) octane (Fluka, Neu-Ulm, Germany) and examined using a confocal laser scanning microscope (LSM 710 combined with Axio Observer; Carl Zeiss, Oberkochen, Germany). For quantification, the fraction of cells showing a perinuclear accumulation of BACE1 was quantified from at least 30 cells per clone from three independent experiments using 0.9-lm thick maximum intensity sections (Fig. 3). The data are shown as a percentage of cells showing a perinuclear BACE1 accumulation. A region of interest was defined as the region showing LAMP3 staining and the Pearson coefficient for colocalization of BACE1 and LAMP3 in this region of interest was determined (Fig. 4). At least 30 cells per clone from three different experiments were scored. A JACoP Plug-in for IMAGEJ (NIH, Bethesda, MD, USA) was used for quantification [48]. As a result of background staining, a threshold level was defined for each experiment (i.e. the same threshold for all cell clones within one experiment) and the total fluorescent signals for BACE1 were determined using 0.9 lm thick maximum intensity confocal sections (Fig. 5). At least 30 cells per clone from three individual experiments were measured. Statistical analysis was carried out by one-way analysis of variance with a Bonferroni post-hoc test. Bar graphs show the mean
SD. P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) were considered statistically significant.

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HeLa cells grown on coverslips were fixed in methanol and a PLA was performed using the Duolink kit (SigmaAldrich) with orange detection reagents, in accordance with the standard protocol and using the reagents provided by the manufacturer. Flotillin-1 and flotillin-2 were detected with polyclonal rabbit antibodies (both from Sigma-Aldrich; 1/100 dilution for flotillin-1 and 1/200 dilution for flotillin2) and BACE1 was detected with the mouse monoclonal antibody (diluted to 10 lgmL–1; a gift from W. Annaert). For negative controls, one of the primary antibodies was left out or the staining was performed with flotillin-2 knockdown cells. Quantification was performed by counting the mean number of positive signals per cell from four independent experiments (~ 300 cells/labeling/experiment).

Densitometry and statistical analysis Immunoblots were scanned and densitometric analysis was performed using QUANTITY ONE (Bio-Rad, Hercules, CA, USA). Intensity values were corrected for background and normalized as stated. Analysis of variance was performed using GRAPHPAD PRISM, version 5.0 (GraphPad Software Inc., San Diego, CA, USA). Unless otherwise stated, all experiments were performed at least three times. P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) were considered statistically significant.

Electronic manipulation of the images The presented images have in some cases been subjected to contrast or brightness adjustments. No other manipulations were performed unless stated otherwise.

Acknowledgements This work has been supported by the Alzheimer Forschung Initiative e.V. (Grant number 07809), Germany; by a research grant of the University Medical Center Giessen and Marburg (UKGM, Grant FEBS Journal 281 (2014) 2074–2087 ª 2014 FEBS

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number 4/2010 GI); and by the Deutsche Forschungsgemeinschaft (DFG, Grant Ti291/6-2). We thank Professor W. Kummer for allowing us to use the microscope facility of the Institute of Anatomy and Cell Biology, Giessen. G. Tesco is acknowledged for providing us with the BACE1-myc expression constructs and B. De Strooper and W. Annaert are thanked for the kind gift of the BACE1 antibody. We thank Petra Janson and Ralf F€ ullkrug for their skillful technical assistance. The authors declare that there are no conflicts of interest. BAJ carried out the GST pulldown and APP processing experiments and prepared samples for microscopy. MM was responsible for the confocal microscopy and performed the quantitative analysis. AB analyzed the BACE1 expression upon stable flotillin knockdown. RT cloned the GST fusion constructs, performed the immunoprecipitation and PLA experiments, and generated the stable flotillin knockdown cells. BAJ, MM and AB drafted the figures and RT made the final versions. RT was responsible for the design and supervision of the study. All authors participated in the writing of the manuscript, and read and approved the final version submitted for publication.

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Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site: Fig. S1. Flotillin depletion does not influence the subcellular localization of BACE1-myc LLAA. Fig. S2. Enhanced localization of BACE1-myc in late endosomes upon flotillin knockdown. Fig. S3. No colocalization of BACE1 with the early endosomal marker Rab5 or Golgi marker Golgin-97 in control and flotillin knockdown cells.

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