Brox, a novel farnesylated Bro1 domain-containing protein that associates with charged multivesicular body protein (CHMP4) Fumitaka Ichioka, Ryota Kobayashi, Keiichi Katoh, Hideki Shibata and Masatoshi Maki Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Japan Keywords Alix; Bro1; CHMP4; ESCRT-III; farnesylation Correspondence M Maki, Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan Fax: +81 52 789 5542 Tel: +81 52 789 4088 E-mail: mmaki@agr.nagoya-u.ac.jp Database The nucleotide sequence of the human Brox cDNA is available in the DDBJ ⁄ EMBL ⁄ GenBank database under accession number AB276123 (Received 11 October 2007, revised December 2007, accepted 10 December 2007) doi:10.1111/j.1742-4658.2007.06230.x Human Brox is a newly identified 46 kDa protein that has a Bro1 domainlike sequence and a C-terminal thioester-linkage site of isoprenoid lipid (CAAX motif) (C standing for cysteine, A for generally aliphatic amino acid, and X for any amino acid) Mammalian Alix and its yeast ortholog, Bro1, are known to associate with charged multivesicular body protein (CHMP4), a component of endosomal sorting complex required for transport III, via their Bro1 domains and to play roles in sorting of ubiquitinated cargoes We investigated whether Brox has an authentic Bro1 domain on the basis of its capacity for interacting with CHMP4s Both Strep Tactin binding sequence (Strep)-tagged wild-type Brox (Strep–BroxWT) and Strep-tagged farnesylation-defective mutant (Cys fi Ser mutation; Strep– BroxC408S) pulled down FLAG-tagged CHMP4b that was coexpressed in HEK293 cells Treatment of cells with a farnesyltransferase inhibitor, FTI277, caused an electrophoretic mobility shift of Strep–BroxWT, and the mobility coincided with that of Strep–BroxC408S The inhibitor also caused a mobility shift of endogenous Brox detected by western blotting using polyclonal antibodies to Brox, suggesting farnesylation of Brox in vivo Fluorescence microscopic analyses revealed that Strep–BroxWT exhibited accumulation in the perinuclear area and caused a punctate pattern of FLAG–CHMP4b that was constitutively expressed in HEK293 cells On the other hand, Strep–BroxC408S showed a diffuse pattern throughout the cell, including the nucleus, and did not cause accumulation of FLAG– CHMP4b Fluorescent signals of monomeric green fluorescent protein (mGFP)-fused BroxWT merged partly with those of Golgi markers and with those of abnormal endosomes induced by overexpression of a dominant negative mutant of AAA type ATPase SKD1 ⁄ Vps4B in HeLa cells, but such colocalization was less efficient for mGFP–BroxC408S These results suggest a physiological significance of farnesylation of Brox in its subcellular distribution and efficient interaction with CHMP4s in vivo Alix (also named AIP1) is an interacting partner of the penta-EF-hand Ca2 + -binding protein, ALG-2 [1–5], and acts as a multifunctional adaptor protein in vari- ous cellular functions such as cell death, receptor endocytosis, endosomal protein sorting, cell adhesion, budding of enveloped RNA viruses and development Abbreviations BroxWT, wild-type Brox; CAAX motif, a thioester-linkage site of isoprenoid lipid (C standing for cysteine, A for generally aliphatic amino acid, and X for any amino acid); CHMP, charged multivesicular body protein; E-64, trans-epoxysuccinyl-L-leucylamido(4-guanidino) butane; ESCRT, endosomal sorting complex required for transport; FTI-27, farnesyltransferase inhibitor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GST, glutathione S-transferase; mGFP, monomeric green fluorescent protein; pAb, polyclonal antibody; PTP, protein tyrosine phosphatase; PVDF, poly(vinylidene difluoride); RabGAPLP, Rab GTPase-activating protein-like protein; Strep, StrepTactin binding sequence 682 FEBS Journal 275 (2008) 682–692 ª 2008 The Authors Journal compilation ª 2008 FEBS F Ichioka et al [6–9] Alix associates through its Pro-rich region with SH3 domains of CIN85 ⁄ Rukl ⁄ SETA [10], endophilins [11] and Src [12] at sites different from the ALG-2 binding site [13] The PSAP sequence present in the Pro-rich region of Alix is also recognized by the ubiquitin E2 variant domain of TSG101 [14] The crystal 3D structures of the N-terminal domain (designated Bro1 domain) of yeast Bro1 [15] and that of human Alix [16] have revealed folded cores of  360 residues The Bro1 domains are necessary and sufficient for binding to endosomal sorting complex required for transport (ESCRT)-III component charged multivesicular body protein (CHMP4) ⁄ Snf7 [16–18] ESCRTs and their associated proteins are conserved from yeast to humans, and function in the sorting of ubiquitinated cargoes into intraluminal vesicles that are generated by inward budding of the endosomal membrane of the so-called multivesicular bodies (endosomes) [19,20] A V domain in the central region of Alix associates with the YPXnL motif found in late domains of retroviral Gag proteins [16,21] and it is involved in regulation of viral budding from cells Two Alix homologs containing Bro1 domain-like sequences are found in the human genome database (Fig 1) One is PTPN23, which encodes a putative protein tyrosine phosphatase, HD-PTP [22] We previously demonstrated that, as in the case of Alix, HD-PTP interacts not only with CHMP4b through its N-terminal region containing a Bro1 domain-like sequence but also with TSG101, endophilin A1 and ALG-2 through its central Pro-rich region, indicating that HD-PTP is a functional paralog of Alix [23] The second Alix homolog, designated Brox [a Bro1 domain-containing protein with a thioester-linkage site of isoprenoid lipid (CAAX motif, C standing for cysteine, A for generally aliphatic amino acid, and X for any amino acid)] in this article, is a 411 amino acid Association of Brox with CHMP4 residue hypothetical protein encoded by C1orf58 (FLJ32421), which was first reported as one of 295 proteins identified in exosomes (extracellular microvesicles) in human urine [24] Brox lacks a V domain and Pro-rich region Multiple sequence alignment and phylogenetic analysis of Alix orthologs and Bro1 domain-containing proteins have revealed that the Bro1 domain-like sequence of Brox is less similar to the Bro1 domains of Alix and HD-PTP and even more distantly related than yeast Bro1 (supplementary Fig S1) It has remained to be established whether Brox has a functional Bro1 domain, i.e a capacity for binding to CHMP4s A unique feature of Brox is that it possesses a C-terminal tetrapeptide sequence Cys-Tyr-Ile-Ser, which conforms to a CAAX motif [25] In this study, by expressing in cultured mammalian cells an epitope-tagged wild-type Brox as well as its amino acid-substituted mutant in the CAAX motif, we investigated the capacity of Brox to bind to CHMP4s We also investigated the subcellular distribution of Brox by confocal fluorescence microscopy and the extracellular release of Brox into culture medium The results suggest that Brox has a functional Bro1 domain for CHMP4 interaction Although farnesylation is dispensable for in vitro interaction and extracellular release, post-translational lipid modification facilitates interaction with CHMP4s in vivo by restricting its subcellular localization Results Conservation of Brox in the animal kingdom Orthologs of human Brox are found in the animal kingdom, including Caenorhabditis elegans, but there is no ortholog in the currently available Drosophila genome database (FlyBase) Brox-like genes are not Fig Schematic representations of human Bro1 domain-containing proteins Schematic representations of Alix, HD-PTP and Brox are shown Bro1, Bro1 domain; V, V domain, PRR, Pro-rich region; PTP, protein tyrosine phosphatase domain; PEST, PEST motif; CAAX, CAAX motif The numbers indicate the amino acid residues Cys408 was substituted with Ser for creation of a farnesylation-defective mutant in this study FEBS Journal 275 (2008) 682–692 ª 2008 The Authors Journal compilation ª 2008 FEBS 683 Association of Brox with CHMP4 F Ichioka et al found in plants, fungi or other lower eukaryotes The C-terminal CAAX-motif-like tetrapeptide sequences are conserved except for the first A (human, Tyr; mouse, Ser; chicken, Arg; C elegans, Val) (supplementary Fig S2), which is not necessarily aliphatic for the prenylation motif [25] A Cys residue in a CAAX motif is covalently linked with either a 15-carbon isoprenoid, catalyzed by farnesyltransferase, or a 20-carbon isoprenoid, catalyzed by geranylgeranyltransferase I Brox has a preferred sequence for farnesyltransferase, due to the presence of the C-terminal Ser residue [25,26] A B Interaction of Brox with CHMP4b Whereas CHMP4s interact with both Alix [14,17,18,27–29] and HD-PTP [29], Rab GTPase-activating protein-like protein (RabGAPLP) interacts with Alix but not with HD-PTP [29] We investigated whether Brox interacts with CHMP4s and RabGAPLP Lysates of HEK293 cells coexpressing either wild-type StrepTactin binding sequence (Strep)-tagged Brox (Strep–BroxWT) or Strep-tagged CAAX motif mutant (substitution of Cys408 with Ser; Strep–BroxC408S) and either FLAG–CHMP4b or FLAG–RabGAPLP were incubated with StrepTactin sepharose beads, and the complexes were pulled down Then, the proteins bound to the beads (pulldown products) were subjected to western blotting using mAb to FLAG FLAG– CHMP4b was pulled down with both Strep–BroxWT and Strep–BroxC408S and with the comparable N-terminal regions containing the Bro1 domain of Alix (Strep– Alix1)423) and HD-PTP (Strep–HD-PTP1)431) (Fig 2A) On the other hand, FLAG–RabGAPLP was pulled down with Strep–Alix1)424 but not with either Brox or HD-PTP constructs (Fig 2B) Effects of farnesylation inhibition on electrophoretic mobility of Brox As shown in Fig 2, the CAAX-motif mutant Strep– BroxC408S exhibited retardation of migration in SDS ⁄ PAGE To determine whether the difference in migration rates between the wild-type and the mutant is related to the potential farnesylation at Cys408, a farnesyltransferase inhibitor, FTI-277 [30], was added to the culture medium during transient overexpression of Strep-tagged Brox proteins Treatment of cells with FTI-277 caused a shift in the mobility of Strep–BroxWT to that of Strep–BroxC408S, but the treatment did not affect that of Strep–BroxC408S (Fig 3A) Addition of the inhibitor to the culture medium also caused an electrophoretic mobility shift of endogenous Brox that was 684 Fig Brox interacts with CHMP4b but not with RabGAPLP HEK293 cells were cotransfected with pFLAG–CHMP4b (A) or pFLAG–RabGAPLP (B) and either an empty vector of Strep-tag (pEXPR-IBA105) (Ctrl), pStrep–BroxWT, pStrep–BroxC408S, pStrep– Alix1)423 or pStrep–HD-PTP1)431 The cleared cell lysates (Input) were incubated with StrepTactin sepharose beads at °C overnight The pellets (Pulldown) were subjected to SDS ⁄ PAGE and western blotting PVDF membranes were probed with mAb to FLAG (A and B, upper panels) and mAb to Strep-tag II (A and B, lower panels) detected with polyclonal antibody (pAb) to Brox (Fig 3B) We therefore conclude that Brox is farnesylated at Cys408 in vivo Next, we performed biochemical subcellular fractionation of endogenous Brox in HEK293 cells by differential centrifugation and analysis by western blotting with pAb to Brox As shown in Fig 3C, although samples of the P1, P2 and P3 fractions were loaded 20-fold more than those of the total fraction (T) and cytosolic fraction (S), intensities of the observed immunoreactive signals were similar between P2 and S, and the signals for P1 and P3 were weaker than the signals for S, suggesting that less than  10% of Brox bound to membranes There was no significant difference in the fractionation pattern between farnesylated and unmodified Brox proteins with treatment with FTI-277 Subcellular distribution of Strep–Brox Subcellular distributions of transiently expressed proteins of Strep–BroxWT and Strep–BroxC408S in HEK293 cells were analyzed by confocal immunofluorescence microscopy, using mAb to Strep, and chromosomal DNAs were stained with TO-PRO-3 As shown in Fig 4, Strep–BroxWT (Fig 4A) exhibited a diffuse pattern but some accumulation in the FEBS Journal 275 (2008) 682–692 ª 2008 The Authors Journal compilation ª 2008 FEBS F Ichioka et al A Association of Brox with CHMP4 A B C D E F G H B C Fig Farnesylation of Brox (A) HEK293 cells were transfected with pStrep–BroxWT or pStrep–BroxC408S and cultured in the presence (+) or absence (–) of 10 lM FTI-277 for 24 h The cleared cell lysates were incubated with Strep-tactin sepharose beads at °C for h The pellets (Pulldown) were subjected to SDS ⁄ PAGE and western blotting PVDF membranes were probed with mAb to Strep-tag II (B) HEK293 cells were cultured in the presence (+) or absence ()) of 10 lM FTI-277 for 24 h The total cell lysates were subjected to SDS ⁄ PAGE and western blotting PVDF membranes were probed with pAb to Brox Arrowheads indicate non-specific bands (C) Effects of treatment with FTI-277 on the subcellular distribution of Brox were investigated by subcellular fractionation as described in Experimental procedures T, total lysate; P1, nuclear and cell debris (pellet, 1000 g for min); P2, crude mitochondrial and organelle-enriched fraction (pellet, 10 000 g for 15 min); P3, microsomal fraction (pellet, 100 000 g for 30 min); S, soluble fraction (supernatant, 100 000 g for 30 min) Different relative amounts of fractionated samples (% corresponding to harvested cells: T and S, 0.1%; P1, P2 and P3, 2%) were loaded and analyzed by SDS ⁄ PAGE and immunoblotting analysis with antibody against Brox Arrowheads indicate nonspecific crossreacting bands perinuclear area On the other hand, Strep–BroxC408S (Fig 4C) exhibited a diffuse pattern throughout the cytoplasm and nucleus This diffuse pattern is in contrast to the absence of conspicuous fluorescence signals in nuclei of cells transfected with expression plasmids of Strep–BroxWT (Fig 4A), Strep–Alix1)423 (Fig 4E) and Strep–HD-PTP1)431 (Fig 4G) We previously showed that the distribution of FLAG–CHMP4b that was constitutively expressed in HEK293 cells was diffuse, but that coexpression with C-terminally deleted Alix (AlixDC-V5) caused accumulation of FLAG–CHMP4b in the perinuclear Fig Subcellular distribution of Brox HEK293 cells were transfected with pStrep–BroxWT (A, B), pStrep–BroxC408S (C, D), pStrep– Alix1)423 (E, F) or pStrep–HD-PTP1)431 (G, H) After 24 h, the cells were fixed and stained with mAb to Strep-tag II and Alexa Fluor 488-conjugated goat anti-(mouse IgG) (A, C, E, G) and with TO-PRO-3 for chromosomal DNA (B, D, F, H) The fluorescence signals of Alexa Fluor 488 and TO-PRO-3 were analyzed with a confocal laser-scanning microscope Bars, 10 lm Asterisks indicate transfected cells area [17], and this effect was also observed after coexpression with Strep–Alix1)423 but not with Strep–HDPTP1)431, suggesting a qualitative difference between the Bro1 domains of Alix and HD-PTP in vivo [23] As FEBS Journal 275 (2008) 682–692 ª 2008 The Authors Journal compilation ª 2008 FEBS 685 Association of Brox with CHMP4 F Ichioka et al A B C A B C D E F D E F G H I G H I J K L J K L Fig Formation of CHMP4b puncta by Brox overexpression FLAG–CHMP4b ⁄ HEK293 cells were transfected with pStrep–BroxWT (A–C), pStrep–BroxC408S (D–F), pStrep–Alix1)423 (G–I) or pStrep–HD-PTP1)431 (J–L) After 24 h, the cells were fixed and stained with primary antibodies (mAb to Strep-tag II and pAb to FLAG) and secondary antibodies [Alexa Fluor 488-conjugated goat anti-(mouse IgG) and Cy3-labeled goat anti-(rabbit IgG)] The fluorescence signals of Alexa Fluor 488 (A, D, G, J) (green) and Cy3 (B, E, H, K) (red) were analyzed with a confocal laser-scanning microscope and are represented in black and white The merged images are shown in (C), (F), (I) and (L), respectively, in color Bars, 10 lm Fig Subcellular distribution of Brox HEK293 cells were transfected with pmGFP–BroxWT After 24 h, the cells were fixed and stained with mAbs of organelle markers: anti-GM130 (cis-Golgi), anti-p230 (trans-Golgi), anti-EEA1 (early endosomes) and antiLAMP-1 (late endosomes and lysosomes) Cy3-labeled goat anti(mouse IgG) was used as a secondary antibody The fluorescence signals of mGFP (A, D, G, J) (green) and Cy3 (B, E, H, K) (red) were analyzed with a confocal laser-scanning microscope and represented in black and white The merged images are shown in (C), (F), (I) and (L), respectively, in color The small boxed areas are magnified in the respective large boxed areas Bars, 10 lm shown in Fig 5, qualitative differences in Bro1 domains were also observed among the coexpressed Bro1 domain constructs As in the case of Strep–Alix1)423 (Fig 5G– I), expression of Strep–BroxWT caused accumulation of FLAG–CHMP4b and partial colocalization in the perinuclear area (Fig 5A–C) This accumulation was not observed in cells expressing Strep–BroxC408S (Fig 5D–F) or Strep–HD-PTP1)431 (Fig 5J–L) or in untransfected cells in the same microscopic fields expressed a Brox protein fused with mGFP (mGFP– BroxWT) As shown in Fig 6, a proportion of the fluorescence signals of mGFP–BroxWT expressed in HEK293 cells merged well with those of Golgi marker proteins GM130 (Fig 6A–C) and p230 (Fig 6D–F) No merged signals were observed for an early endosome marker (EEA1) (Fig 6G–I) or a late endosome ⁄ lysosome marker (LAMP-1) (Fig 6J–L) Overexpression of an ATPase-defective mutant of AAA type ATPase SKD1 (also named Vps4B), SKD1E235Q, is known to induce formation of abnormal endosomes As shown in Fig 7, a subset of punctate fluorescence signals of mGFP–BroxWT expressed in HeLa cells merged with those of Myc–SKD1E235Q (Fig 7A–C) A large proportion of mGFP–BroxC408S showed a diffuse pattern, but some fine punctate Analyses of monomeric green fluorescent protein (mGFP)–Brox distribution with organelle markers Next, for further analyses of the perinuclear distribution of Brox by immunostaining with commercially available mAbs against organelle markers, we 686 FEBS Journal 275 (2008) 682–692 ª 2008 The Authors Journal compilation ª 2008 FEBS F Ichioka et al Association of Brox with CHMP4 A B C D E F A B E235Q Fig Colocalization of Brox with SKD1 HeLa cells were cotransfected with pMyc–SKD1E235Q and either with pmGFP–BroxWT (A–C) or with pmGFP–BroxC408S (D–F) After 24 h, cells were fixed and stained with mAb to c-Myc and Cy3-labeled goat anti-(mouse IgG) The fluorescence signals of mGFP (A and D, green) and Cy3 (B and E, red) were analyzed with a confocal laser-scanning microscope and are represented in black and white The merged images are shown in (C) and (F), respectively The small boxed areas are magnified in the respective large boxed areas Bars, 10 lm patterns were also observed (Fig 7D–F) The merging of signals between mGFP–BroxC408S and Myc– SKD1E235Q was much less noticeable than in the case of mGFP–BroxWT Detection of Brox in extracellular vesicles Pisitkun et al [24] performed proteomic profiling of extracellular microvesicles (exosomes) in human urine by MS analysis and identified 295 proteins, including a hypothetical protein FLJ32421 (designated Brox in this study) To gain more insights into the release of Brox in exosomes in conjunction with farnesylation, we performed western blotting of vesicles released from HEK293T cells into culture medium As shown in Fig 8A, Brox, Alix and TSG101 (a positive control) were detected with their respective antibodies in 100 000 g pellets (vesicular fraction) of cultured medium that had been precleared by centrifugation at 10 000 g (culture supernatant) but not in the case of the medium without culture (medium) In contrast, glyceraldehyde3-phosphate dehydrogenase (GAPDH), a cytosolic protein used as a negative control, was not detected in the vesicular fraction Similarly, 100 000 g pellets of the culture supernatant from HEK293T cells expressing either FLAG–BroxWT or FLAG–BroxC408S were analyzed by western blotting with mAb to FLAG The intensities of the detected bands were not significantly different between FLAG–BroxWT and FLAG– BroxC408S (Fig 8B) We therefore conclude that Brox Fig Release of Brox from cells (A) HEK293T cells were incubated at 37 °C for 48 h in culture medium containing 10% fetal bovine serum, and vesicles released into the medium were collected by ultracentrifugation as described in Experimental procedures The total cell lysate (lysate) and vesicular fractions were analyzed by western blotting with antibodies against Brox, Alix, TSG101 and GAPDH Cult Sup, cultured medium supernatant; medium, control medium that was incubated without cells (B) HEK293T cells were transfected with pFLAG–BroxWT or pFLAG– BroxC408S After 48 h, transfectants were harvested, and vesicles released from transfectants were collected by ultracentrifugation The total cell lysates (Lysate) and vesicle fractions (vesicles) were analyzed by western blotting with mAb to FLAG is packaged into exosomal vesicles and released to the extracellular milieu but that this process does not depend on its farnesylation Discussion In the human genome database, there are three genes that encode Bro1 domains: Alix, HD-PTP and Brox (Fig 1) Alix and HD-PTP, possessing similarities in a wider region, have been shown to share several associated proteins, such as CHMP4b, TSG101, endophilin A1 and ALG-2 [23] On the other hand, Brox lacks a V domain and Pro-rich region In the present study, we demonstrated for the first time that Brox also associates with CHMP4b (Fig 2A) In our previous study, we found that the Bro1 domain of Alix directly bound the three CHMP4 isoforms (CHMP4a, CHMP4b and CHMP4c), by in vitro GST-pulldown assays using each recombinant protein, and that CHMP4b is a major Alix-interacting isoform, based on their expression levels and binding capacities [18] In the present study, FEBS Journal 275 (2008) 682–692 ª 2008 The Authors Journal compilation ª 2008 FEBS 687 Association of Brox with CHMP4 F Ichioka et al we similarly prepared thioredoxin-fused CHMP4s and glutathione–sepharose beads immobilizing glutathione S-transferase (GST)-fused Brox protein that was purified from Escherichia coli GST–Brox beads pulled down all thioredoxin–CHMP4s (data not shown), indicating direct physical interaction between Brox and CHMP4s Although the physiological significance remains to be established, we reported that the N-terminal domain of Alix associated with RabGAPLP [29], which was later reported to be identical to a Rab5-specific GAP (RabGAP-5) [31] No binding of either Brox or HD–PTP to RabGAPLP was detected, and interaction with RabGAPLP was found to be specific for Alix among the three human Bro1 domain-containing proteins (Fig 2B) Brox contains a C-terminal tetrapeptide motif known as a CAAX motif, which is a site for posttranslational modification with isoprenoids (prenylation) [25] Recently, Maurer-Stroh et al [32], who constructed PRENbase-Database of Prenylated Proteins (http://mendel.imp.ac.at/sat/PrePS/index2.html), obtained experimental evidence, by using 3H-labeled prenyl precursors in an in vitro transcription–translation system, that GST–FLJ32421 is farnesylated but not geranylgeranylated We observed differences in the migration rates in SDS ⁄ PAGE between BroxWT and BroxC408S mutant (Figs and 3A) and between endogenous Brox proteins from farnesyltransferase inhibitor (FTI-277)-treated and untreated cells (Fig 3B) The effect of FTI-277 is smaller on endogenous Brox (Fig 3B) than on transiently expressed Strep–BroxWT (Fig 3A) In these experiments, HEK293 cells were treated with FTI-277 for 24 h However, this length of treatment may not be long enough to replace all pre-existing farnesylated Brox with de novo synthesized unfarnesylated Brox (Fig 3B) On the other hand, the majority of Strep–Brox was synthesized during the period of this inhibitor treatment, and thus only unfarnesylated protein could be detected (Fig 3A) Faster migration of farnesylated proteins than that of unmodified proteins has been reported [33] The electrophoretic mobility shift is explained by sequential enzymatic processing of CAAX proteins after prenylation [25,34]: (a) proteolytic cleavage of the C-terminal AAX residues by Ras-converting enzyme 1; and (b) methylesterification of prenylated Cys by isoprenylcysteine carboxymethyltransferase In addition to the CAAX motif, a second signal is known to be required for Ras proteins to be stably anchored to plasma membranes: palmitoylation of Cys immediately upstream (2–6 residues) of farnesylated Cys of H-Ras, N-Ras and K-Ras4A, and a stretch of basic residues upstream of the farnesylated Cys186 of K-Ras4B [34–36] Brox 688 does not possess immediate upstream Cys residues or a typical basic stretch upstream of the farnesylated Cys408 This fact may explain why only  10% of Brox was recovered in the particulate fractions of HEK293 cells by the biochemical subcellular fractionation, which may release loosely bound Brox from membranes As no significant difference was observed in the distribution pattern between the cells treated and untreated with the farnesyltransferase inhibitor FTI277 (Fig 3C), unmodified Brox may also bind to membranes indirectly by interacting with other proteins Although both Strep–BroxWT and Strep–BroxC408S mutant bound to CHMP4b in the pulldown assay (Fig 2A), a significant difference was observed in the subcellular localization of transiently overexpressed tagged proteins (Fig 4) In contrast to the distribution of Strep–BroxWT, Strep–BroxC408S exhibited a diffuse pattern throughout the cell, including the nucleus This mutational effect of Cys408 agrees with the results of a study by Maurer-Stroh et al showing that the perinuclear condensed distribution of wild-type GFP– FLJ32421 was changed to a diffuse pattern throughout the cell, including the nucleus, by substituting Cys408 with Ala [32] In addition, we observed a difference between the wild-type and the C408S mutant in the capacity to induce accumulation of constitutively expressed FLAG–CHMP4b in HEK293 cells (Fig 5) Moreover, localization of mGFP–BroxWT to abnormal endosomes was induced by overexpression of Myc– SKD1E235Q, but this occurred to a lesser degree in the case of mGFP–BroxC408S both in HeLa cells (Fig 7) and in HEK293 cells (supplementary Fig S3) Interestingly, mGFP–BroxWT showed partial colocalization with cis-Golgi marker protein GM130 (Fig 6A–C) and trans-Golgi marker protein p230 (Fig 6D–F) Whereas farnesylation is catalyzed by cytosolic farnesyltransferase, both post-prenylation processing enzymes, Ras-converting enzyme and isoprenylcysteine carboxymethyltransferase, are endoplasmic reticulum-integral membrane proteins, and proteolytic cleavage and methylesterification are catalyzed on the cytosolic side of the endoplasmic reticulum membrane [34–36] Some processed proteins, either further palmitoylated or not, are transported to the Golgi apparatus and then to the plasma membrane or to other intracellular membranes [35] Thus, it remains to be established whether the observed localization of mGFP-fused Brox to Golgi represents a reservoir of the transiently overexpressed protein that awaits transportation to its final destination, e.g to multivesicular endosomes It would be interesting to investigate whether there exist cytosolic factors that regulate the distribution of farnesylated Brox FEBS Journal 275 (2008) 682–692 ª 2008 The Authors Journal compilation ª 2008 FEBS F Ichioka et al Among the CHMP family members (ESCRT-III components and related proteins containing SNF7 domains), Alix is known to interact with only CHMP4s We investigated whether Brox has specificities different from those of Alix toward CHMPs, each of which has a unique feature, e.g myristoylation (CHMP6) [37], tandem repeat of SNF7-like domain (CHMP7) [38], and binding to phosphatidylinositol 3,5-bisphosphate (CHMP3) [39] We performed a yeast two-hybrid assay using GAL4-DNA-binding domainfused Brox as bait and GAL4-activation domain-fused CHMPs (CHMP1A, CHMP1B, CHMP2A, CHMP2B, CHMP3, CHMP4a, CHMP4b, CHMP4c, CHMP6, CHMP7) as prey Positive interaction was observed only for CHMP4s (data not shown) Other ESCRTrelated proteins, including TSG101, Vps28, Vps37A, EAP20, EAP30, EAP45 and Alix, showed no interactions At present, the physiological function of Brox is not known Secretion of this protein to the extracellular space by exosomes may not represent a bona fide role, because a large number of multivesicular bodyrelated proteins are also secreted [24] We expected that inhibition of farnesylation would reduce secretion of Brox, but there was no significant difference in the amount of secreted Brox proteins between the wildtype and the farnesylation-defective C408S mutant of FLAG-tagged Brox (Fig 8B) This may be explained by their similar capacities for binding to CHMP4b (Fig 2A) The finding of the ability of Brox to bind to a specific ESCRT-III component extends our understanding of the molecular mechanism underlying the recognition of CHMP4 by Bro1 domains Experimental procedures Antibodies and reagents Mouse mAbs were Strep-tag II (IBA GmbH, Gottingen, ¨ Germany), FLAG-tag (M2), c-Myc-tag (9E10) (Sigma, St Louis, MO, USA), GM130, p230 trans-Golgi, CD107a ⁄ Lamp-1, EEA1 (BD Biosciences, San Jose, CA, USA), GAPDH (Chemicon ⁄ Millipore, Billerica, MA, USA), and TSG101 (4A10) (GeneTex, San Antonio, TX, USA) Rabbit pAbs of FLAG-tag and GST were purchased from Sigma and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively Rabbit pAbs against Brox were raised by the conventional method using a GST-fused Brox protein as an antigen and affinity-purified by a column immobilizing maltose-binding protein (MBP)-fused Brox Peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG were obtained from Wako (Osaka, Japan) Preparation of rabbit pAbs against Alix has been described previously [40] Cy3-labeled anti-mouse or rabbit IgG and Alexa Fluor Association of Brox with CHMP4 488-conjugated anti-mouse IgG used for indirect immunofluorescence analyses were obtained from Amersham (Little Chalfont, UK) and BD Biosciences, respectively The following reagents were purchased: farnesyltransferase inhibitor FTI-277 (EMD ⁄ Calbiochem, San Diego, CA, USA), poly(l-lysine) (Sigma) and TO-PRO-3 (Invitrogen ⁄ Molecular Probes, Carlsbad, CA, USA) Construction of plasmids A Brox cDNA (DDBJ accession number: AB276123) was cloned from an HeLa cDNA library by the PCR method, using a pair of primers based on the registered sequence NM_144695 for C1orf58: 5¢-GGG AAT TCA TGA CCC ATT GGT TTC ATA GGA ACC-3¢ and 5¢-GGG AAT TCT TAG GAG ATG TAG CAC CCA GTG TC-3¢ (nucleotides corresponding to a cDNA of the hypothetical protein C1orf58 are underlined), and an EcoRI fragment was inserted into pEXPR-IBA105-A [38] (pStrep–BroxWT), pCMV3 · FLAG-A [17] (pFLAG–BroxWT) and pmGFPC2 (pmGFP–BroxWT), respectively pmGFP-C2, a mammalian expression vector for monomeric enhanced GFP fusion protein [41], was created from pEGFP-C2 (Clontech) by PCR-based site-directed mutagenesis according to the instructions provided with a QuikChange Site-Directed Mutagenesis Kit from Stratagene using two complementary primers (5¢-CAG TCC AAG CTG AGC AAA GAC CCC AAC GAG AAG CGC GAT CAC-3¢ and 5¢-GTG ATC GCG CTT CTC GTT GGG GTC TTT GCT CAG CTT GGA CTG-3¢) pmGFP–Brox C408S , which has a point mutation at amino acid 408, was created by PCR-based site-directed mutagenesis using pmGFP–Brox as a template and complementary primers (5¢-CAA AAG GAC ACT GGG TCC TAC ATC TCC TAA G-3¢ and 5¢-CTT AGG AGA TGT AGG ACC CAG TGT CCT TTT G-3¢) To create pStrep–BroxC408S and pFLAG– BroxC408S, an EcoRI fragment of BroxC408S derived from pmGFP–BroxC408S was inserted into the EcoRI site of pEXPR-IBA105-A and pCMV3 · FLAG-A, respectively pEXPR-IBA105 was purchased from IBA GmbH Constructions of pStrep–Alix1)423, pStrep–HD-PTP1)431, pFLAG– CHMP4b, pFLAG–RabGAPLP and pMyc–SKD1E235Q have been described previously [17,18,23,29] Cell culture and transfection HEK293 cells were subjected to limiting dilution cloning, and one of the isolated cell lines, designated YS14, was used in this study FLAG–CHMP4b ⁄ HEK293 cells, HEK293 cells constitutively expressing FLAG–CHMP4b, were established as described previously [17] HEK293 YS14, FLAG–CHMP4b ⁄ HEK293 and HeLa cells were cultured in DMEM supplemented with 5% (HEK293 cells) or 10% (HeLa cells) heat-inactivated fetal bovine serum, FEBS Journal 275 (2008) 682–692 ª 2008 The Authors Journal compilation ª 2008 FEBS 689 Association of Brox with CHMP4 F Ichioka et al penicillin (100 unitsỈmL)1) and streptomycin (100 lgỈmL)1) at 37 °C under humidified air containing 5% CO2 One day after cells had been seeded, the cells were transfected with the expression plasmid DNAs by the conventional calcium phosphate precipitation method or by using FuGENE6 (Roche, Basel, Switzerland) Strep-pulldown assay At 24 h after transfection with expression vectors, HEK293 cells were washed and harvested with NaCl ⁄ Pi (137 mm NaCl, 2.7 mm KCl, mm Na2HPO4, 1.5 mm KH2PO4, pH 7.3) and then lysed in lysis buffer A (10 mm Hepes ⁄ NaOH, pH 7.4, 142.5 mm KCl, 0.2% Nonidet P-40, 0.1 mm pefabloc, 25 lgỈmL)1 leupeptin, lm pepstatin and lm E-64) Supernatants after centrifugation at 14 000 r.p.m were incubated with Strep-tactin sepharose (IBA GmbH) at °C overnight with gentle mixing After the beads had been recovered by low-speed centrifugation and washed three times with the lysis buffer without protease inhibitors, the bead-bound proteins (pulldown products) were subjected to SDS ⁄ PAGE followed by western blotting using poly(vinylidene difluoride) (PVDF) membranes (Immobilon-P) (Millipore) The membranes were then blotted either with mAb to Strep-tag II or mAb to FLAG, and then with a horseradish peroxidase-conjugated secondary antibody Signals were detected by the chemiluminescence method using Super Signal West Pico Chemiluminescent Substrate (PIERCE, Rockford, IL, USA) tions were obtained by differential centrifugation (see Fig legend for centrifugation details), essentially as described previously [40] Immunofluorescence microscopic analyses One day after cells had been seeded on coverslips that had been precoated with poly(l-lysine) (HEK293 cells) or uncoated (HeLa cells), they were transfected with the expression plasmid DNAs After 24 h, cells were washed with NaCl ⁄ Pi, fixed in 4% (w ⁄ v) paraformaldehyde in NaCl ⁄ Pi for 20 min, quenched in 50 mm NH4Cl in NaCl ⁄ Pi for 15 min, and permeabilized in 0.1% (w ⁄ v) Triton X-100 in NaCl ⁄ Pi for After blocking with 0.1% (w ⁄ v) gelatin in NaCl ⁄ Pi for 30 min, the cells were incubated with primary antibodies (mAb to Strep-tag II, pAb to FLAG, mAb to GM130, mAb to p230 trans-Golgi, and mAb to c-Myc-tag) at room temperature for h and then with secondary antibodies [Alexa Fluor 488-conjugated goat anti(mouse IgG) and Cy3-labeled goat anti-(mouse IgG) or Cy3-labeled goat anti-(rabbit IgG)] at room temperature for h For chromosomal DNA staining, cells were incubated with 0.2 lm TO-PRO-3 in NaCl ⁄ Pi at room temperature for 15 Finally, they were mounted with antifading solution [25 mm Tris ⁄ HCl (pH 8.7), 10% polyvinyl alcohol, 5% glycerol, 2.5% 1,4-diazobicyclo(2,2,2)-octane], and analyzed under a confocal laser-scanning microscope (LSM5 PASCAL; Carl Zeiss, Oberkochen, Germany) Analyses of extracellular vesicles (exosomes) Treatment with farnesyltransferase inhibitor At h after HEK293 cells had been transfected with expression vectors, the culture medium was changed to DMEM containing 5% fetal bovine serum supplemented with FTI277 to a final concentration of 10 lm or vehicle (0.1% dimethylsulfoxide) After 24 h, cells were washed and harvested with NaCl ⁄ Pi and then lysed and subjected to Strep pulldown as described above, except that cleared lysates were incubated with Strep-tactin sepharose at °C for h For analysis of endogenous Brox, the total cell lysates of HEK293 cells cultured in DMEM containing 5% fetal bovine serum supplemented with FTI-277 to a final concentration of 10 lm were analyzed by western blotting using pAb to Brox Subcellular fractionation After treatment with lm FTI-277 for 48 h, HEK293 cells were suspended in buffer B (10 mm Hepes ⁄ KOH, pH 7.6, 10 mm KCl, 1.5 mm MgCl2, mm 2-mercaptoethanol, 0.1 mm pefabloc, 25 lgỈmL)1 leupeptin, lm E-64, lm pepstatin), freeze–thawed twice, and lysed by passing them 20 times through a 26-gauge needle After addition of NaCl (final concentration 150 mm) to the lysates, subcellular frac- 690 The transfected or untransfected HEK293T cells were incubated in 10% fetal bovine serum ⁄ DMEM for 48 h at 37 °C Then, the culture media were collected and centrifuged at 1000 g and 10 000 g for each to remove cell debris, and the supernatants were further centrifuged at 100 000 g for 30 at °C The pellets were washed by suspending in NaCl ⁄ Pi and recentrifuging under the same conditions The collected vesicles were subjected to western blotting Acknowledgements We thank Ms H Yoshida for subcloning of HEK293 cells and Dr K Hitomi for valuable suggestions This work was supported by a Grant-in-Aid for Scientific Research B (to M Maki), a Grant-in-Aid for Young Scientists B (to H Shibata) and Fellowships for Young Scientists (to F Ichioka) from JSPS References ` Vito P, Lacana E & D’Adamio L (1996) Interfering with apoptosis: Ca2 + -binding protein ALG-2 and FEBS Journal 275 (2008) 682–692 ª 2008 The Authors Journal compilation ª 2008 FEBS F Ichioka et al 10 11 12 13 14 15 Alzheimer’s disease gene ALG-3 Science 271, 521– 525 Vito P, Pellegrini L, Guiet C & D’Adamio L (1999) Cloning of AIP1, a novel protein that associates with the apoptosis-linked gene ALG-2 in a Ca2 + -dependent reaction J Biol Chem 274, 1533–1540 Missotten M, Nichols A, Rieger K & Sadoul R (1999) Alix, a novel mouse protein undergoing calciumdependent interaction with the apoptosis-linked-gene (ALG-2) protein Cell Death Differ 6, 124–129 Maki M, Kitaura Y, Satoh H, Ohkouchi S & Shibata H (2002) Structures, functions and molecular evolution of the penta-EF-hand Ca2+-binding proteins Biochim Biophys Acta 1600, 51–60 Maki M & Shibata H (2007) The penta-EF-hand protein ALG-2 and its interacting ptoteins Calcium Binding Proteins 2, 4–10 Tarabykina S, Mollerup J, Winding P & Berchtold MW (2004) ALG-2, a multifunctional calcium binding protein? Front Biosci 9, 1817–1832 Odorizzi G (2006) The multiple personalities of Alix J Cell Sci 119, 3025–3032 Sadoul R (2006) Do Alix and ALG-2 really control endosomes for better or for worse? Biol Cell 98, 69–77 Mattei S, Klein G, Satre M & Aubry L (2006) Trafficking and developmental signaling: Alix at the crossroads Eur J Cell Biol 85, 925–936 Chen B, Borinstein SC, Gillis J, Sykes VW & Bogler O ă (2000) The glioma-associated protein SETA interacts with AIP1 ⁄ Alix and ALG-2 and modulates apoptosis in astrocytes J Biol Chem 275, 19275–19281 Chatellard-Causse C, Blot B, Cristina N, Torch S, Missotten M & Sadoul R (2002) Alix (ALG-2-interacting protein X), a protein involved in apoptosis, binds to endophilins and induces cytoplasmic vacuolization J Biol Chem 277, 29108–29115 Schmidt MH, Dikic I & Bogler O (2005) Src phosphorylation of Alix ⁄ AIP1 modulates its interaction with binding partners and antagonizes its activities J Biol Chem 280, 3414–3425 Shibata H, Yamada K, Mizuno T, Yorikawa C, Takahashi H, Satoh H, Kitaura Y & Maki M (2004) The penta-EF-hand protein ALG-2 interacts with a region containing PxY repeats in Alix ⁄ AIP1, which is required for the subcellular punctate distribution of the amino-terminal truncation form of Alix ⁄ AIP1 J Biochem 135, 117–128 von Schwedler UK, Stuchell M, Muller B, Ward DM, Chung HY, Morita E, Wang HE, Davis T, He GP, Cimbora DM et al (2003) The protein network of HIV budding Cell 114, 701–713 Kim J, Sitaraman S, Hierro A, Beach BM, Odorizzi G & Hurley JH (2005) Structural basis for endosomal targeting by the Bro1 domain Dev Cell 8, 937–947 Association of Brox with CHMP4 16 Fisher RD, Chung HY, Zhai Q, Robinson H, Sundquist WI & Hill CP (2007) Structural and biochemical studies of ALIX ⁄ AIP1 and its role in retrovirus budding Cell 128, 841–852 17 Katoh K, Shibata H, Suzuki H, Nara A, Ishidoh K, Kominami E, Yoshimori T & Maki M (2003) The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting J Biol Chem 278, 39104–39113 18 Katoh K, Shibata H, Hatta K & Maki M (2004) CHMP4b is a major binding partner of the ALG-2interacting protein Alix among the three CHMP4 isoforms Arch Biochem Biophys 421, 159–165 19 Slagsvold T, Pattni K, Malerød L & Stenmark H (2006) Endosomal and non-endosomal functions of ESCRT proteins Trends Cell Biol 16, 317–326 ´ 20 Williams RL & Urbe S (2007) The emerging shape of the ESCRT machinery Nat Rev Mol Cell Biol 8, 355–368 21 Lee S, Joshi A, Nagashima K, Freed EO & Hurley JH (2007) Structural basis for viral late-domain binding to Alix Nat Struct Mol Biol 14, 194–199 22 Toyooka S, Ouchida M, Jitsumori Y, Tsukuda K, Sakai A, Nakamura A, Shimizu N & Shimizu K (2000) HDPTP: a novel protein tyrosine phosphatase gene on human chromosome 3p21.3 Biochem Biophys Res Commun 278, 671–678 23 Ichioka F, Takaya E, Suzuki H, Kajigaya S, Buchman VL, Shibata H & Maki M (2007) HD-PTP and Alix share some membrane-traffic related proteins that interact with their Bro1 domains or proline-rich regions Arch Biochem Biophys 457, 142–149 24 Pisitkun T, Shen RF & Knepper MA (2004) Identification and proteomic profiling of exosomes in human urine Proc Natl Acad Sci USA 101, 13368– 13373 25 Clarke S (1992) Protein isoprenylation and methylation at carboxyl-terminal cysteine residues Annu Rev Biochem 61, 355–386 26 Moores SL, Schaber MD, Mosser SD, Rands E, O’Hara MB, Garsky VM, Marshall MS, Pompliano DL & Gibbs JB (1991) Sequence dependence of protein isoprenylation J Biol Chem 266, 14603–14610 27 Strack B, Calistri A, Craig S, Popova E & Gottlinger HG (2003) AIP1 ⁄ ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding Cell 114, 689–699 28 Martin-Serrano J, Yarovoy A, Perez-Caballero D & Bieniasz PD (2003) Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins Proc Natl Acad Sci USA 100, 12414–12419 29 Ichioka F, Horii M, Katoh K, Terasawa Y, Shibata H & Maki M (2005) Identification of Rab GTPase-activating protein-like protein (RabGAPLP) as a novel FEBS Journal 275 (2008) 682–692 ª 2008 The Authors Journal compilation ª 2008 FEBS 691 Association of Brox with CHMP4 30 31 32 33 34 35 36 37 38 692 F Ichioka et al Alix ⁄ AIP1-interacting protein Biosci Biotechnol Biochem 69, 861–865 Lerner EC, Qian Y, Blaskovich MA, Fossum RD, Vogt A, Sun J, Cox AD, Der CJ, Hamilton AD & Sebti SM (1995) Ras CAAX peptidomimetic FTI-277 selectively blocks oncogenic Ras signaling by inducing cytoplasmic accumulation of inactive Ras–Raf complexes J Biol Chem 270, 26802–26806 Haas AK, Fuchs E, Kopajtich R & Barr FA (2005) A GTPase-activating protein controls Rab5 function in endocytic trafficking Nat Cell Biol 7, 887–893 Maurer-Stroh S, Koranda M, Benetka W, Schneider G, Sirota FL & Eisenhaber F (2007) Towards complete sets of farnesylated and geranylgeranylated proteins PLoS Comput Biol 3, doi:10.1371/journal.pcbi.0030066 Benetka W, Koranda M, Maurer-Stroh S, Pittner F & Eisenhaber F (2006) Farnesylation or geranylgeranylation? Efficient assays for testing protein prenylation in vitro and in vivo BMC Biochem 7, doi:10.1186/14712091-7-6 Winter-Vann AM & Casey PJ (2005) Post-prenylationprocessing enzymes as new targets in oncogenesis Nat Rev Cancer 5, 405–412 Hancock JF (2003) Ras proteins: different signals from different locations Nat Rev Mol Cell Biol 4, 73–84 Plowman SJ & Hancock JF (2005) Ras signaling from plasma membrane and endomembrane microdomains Biochim Biophys Acta 1746, 274–283 Yorikawa C, Shibata H, Waguri S, Hatta K, Horii M, Katoh K, Kobayashi T, Uchiyama Y & Maki M (2005) Human CHMP6, a myristoylated ESCRT-III protein, interacts directly with an ESCRT-II component EAP20 and regulates endosomal cargo sorting Biochem J 387, 17–26 Horii M, Shibata H, Kobayashi R, Katoh K, Yorikawa C, Yasuda J & Maki M (2006) CHMP7, a novel ESCRT-III-related protein, associates with CHMP4b and functions in the endosomal sorting pathway Biochem J 400, 23–32 39 Whitley P, Reaves BJ, Hashimoto M, Riley AM, Potter BV & Holman GD (2003) Identification of mammalian Vps24p as an effector of phosphatidylinositol 3,5-bisphosphate-dependent endosome compartmentalization J Biol Chem 278, 8786–8795 40 Shibata H, Suzuki H, Yoshida H & Maki M (2007) ALG-2 directly binds Sec31A and localizes at endoplasmic reticulum exit sites in a Ca2 + -dependent manner Biochem Biophys Res Commun 353, 756–763 41 Zacharias DA, Violin JD, Newton AC & Tsien RY (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells Science 296, 913–916 Supplementary material The following supplementary material is available online: Fig S1 Multiple sequence alignment and phylogenetic tree of Bro1-domain-containing proteins Fig S2 Multiple sequence alignment of Brox orthologs Fig S3 Colocalization of Brox with SKD1E235Q in HEK293 cells This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 682–692 ª 2008 The Authors Journal compilation ª 2008 FEBS ... has a point mutation at amino acid 40 8, was created by PCR-based site-directed mutagenesis using pmGFP–Brox as a template and complementary primers (5¢-CAA AAG GAC ACT GGG TCC TAC ATC TCC TAA... AAT TCA TGA CCC ATT GGT TTC ATA GGA ACC-3¢ and 5¢-GGG AAT TCT TAG GAG ATG TAG CAC CCA GTG TC-3¢ (nucleotides corresponding to a cDNA of the hypothetical protein C1orf58 are underlined), and an... Peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG were obtained from Wako (Osaka, Japan) Preparation of rabbit pAbs against Alix has been described previously [40 ] Cy3-labeled anti-mouse