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Genes to Cells (2007) 12, 709-719. doi:10.1111/j.1365-2443.2007.01086.x
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Anchoring of the 26S proteasome to the organellar membrane by FKBP38

 Tadashi Nakagawa1,2, Michiko Shirane1,2,3, Shun-ichiro Iemura4, Tohru Natsume4 and Keiichi I. Nakayama1,2,*

1 Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan
2 CREST, and 3 PRESTO, Japan Science and Technology Corporation (JST), Kawaguchi, Saitama 332-0012, Japan
4 National Institute of Advanced Industrial Science and Technology (AIST), Biological Information Research Center (JBIC), Kohtoh-ku, Tokyo 135-0064, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
FK506-binding protein 38 (FKBP38) is a member of the immunophilin family that resides in the mitochondrial outer membrane and the endoplasmic reticulum (ER) membrane. To investigate the physiological function of FKBP38, we performed a comprehensive search for proteins with which it interacts in human cells by liquid chromatographic and mass spectrometric analysis of FKBP38 immunoprecipitates. Almost all subunits of the 26S proteasome were thus found to interact with FKBP38. In vivo co-immunoprecipitation analyses confirmed that FKBP38 indeed associates with the 26S proteasome via its three tandem tetratricopeptide repeats (TPRs). Binding assays in vitro also revealed that FKBP38 directly interacts with the S4 subunit of the 19S proteasome. Immunofluorescence analysis demonstrated that the subcellular distributions of FKBP38 and the 26S proteasome partially overlapped at mitochondria. Both the abundance and activity of the proteasome in a membrane fraction were markedly reduced for mouse embryonic fibroblasts prepared from Fkbp38–/– mice compared with those prepared from wild-type mice. These results suggest that FKBP38 functions to anchor the 26S proteasome at the organellar membrane.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
FK506-binding protein 38 (FKBP38) is a member of the immunophilin family of proteins that binds to the immunosuppressant drug FK506 (Lam et al. 1995; Pedersen et al. 1999; Shirane & Nakayama 2003). Many FKBPs identified to date share a conserved peptidyl-prolyl isomerase, or FKBP, domain, which is responsible for binding to FK506 (Snyder et al. 1998). In addition to its FKBP domain, FKBP38 contains three tandem tetratricopeptide repeats (TPRs), a calmodulin-binding domain and a transmembrane domain. The TPR is considered to mediate protein–protein interactions (D’Andrea & Regan 2003). Given that FKBP52, whose overall structure is similar to that of FKBP38 but lacks a transmembrane domain, binds to heat shock protein 90 (Hsp90) via its TPR domain (Davies & Sanchez 2005), FKBP38 may also interact with other proteins that are integral to its physiological function. We previously showed that FKBP38 is localized predominantly to the outer membrane of mitochondria as well as endoplasmic reticulum (ER) membrane and that it targets the anti-apoptotic proteins Bcl-2 and Bcl-xL to these organelles, resulting in inhibition of apoptosis (Shirane & Nakayama 2003). In addition, FKBP38 has been shown to stabilize Bcl-2 (Kang et al. 2005). FKBP38 also interacts with presenilins, forming macromolecular complexes together with Bcl-2 (Wang et al. 2005). Presenilins inhibit FKBP38-mediated mitochondrial targeting of Bcl-2 through a {gamma}-secretase-independent mechanism, thereby increasing the susceptibility of cells to apoptosis.

The ubiquitin-proteasome system contributes to many basic cellular processes of eukaryotes, including the cell cycle, the immune response, signal transduction, DNA repair, apoptosis and protein quality control (Hershko et al. 2000; Glickman & Ciechanover 2002). It does so by regulating the stability of protein substrates through polyubiquitin tagging and degradation by the 26S proteasome. The 26S proteasome is an ATP-dependent macromolecular complex (Baumeister et al. 1998; Bochtler et al. 1999; Voges et al. 1999; Kisselev & Goldberg 2001; Pickart & Cohen 2004) that consists of a proteolytic core particle (the 20S proteasome) sandwiched between two 19S regulatory particles. Genetic studies in yeast indicate that the 19S proteasome is responsible for recognition of polyubiquitin-tagged substrates, removal of the polyubiquitin tag from these substrates, substrate unfolding and substrate translocation into the 20S core, steps that require the 19S components Rpn10, Rpn11, Rpt1 to Rpt6 and Rpt2, respectively.

The proteasome is localized both in the cytoplasm and in the nucleus (Rivett 1998; Hirsch & Ploegh 2000; Wojcik & DeMartino 2003). In the cytoplasm, it is associated with several intracellular compartments to fulfill compartment-specific functions, but the mechanisms responsible for such interactions between the proteasome and intracellular compartments are largely unknown (Gorbea et al. 2004; Kalies et al. 2005; Takeda & Yanagida 2005). We have now shown that FKBP38 binds directly via its TPR domain to the S4 subunit of the 19S proteasome. We also found that both the abundance and activity of the proteasome in a membrane fraction were significantly reduced in cells that lack endogenous FKBP38. Overall, these results suggest that FKBP38 tethers the proteasome to the organellar membrane and thereby increases the amount and activity of the proteasome in the membrane fraction.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
FKBP38 interacts with S4 subunit of the 26 proteasome

The immunophilin FKBP38 exhibits chaperone activity and contains three tandem TPRs, a motif considered to mediate protein–protein interaction (Lam et al. 1995; Pedersen et al. 1999; Shirane & Nakayama 2003). To investigate the physiological role of FKBP38, we adopted a comprehensive approach to identify proteins with which it is physically associated in cells. Human FKBP38 tagged with the FLAG epitope at its NH2-terminus was expressed in HEK293T cells and then purified together with associated proteins from cell lysates with an anti-FLAG immunoaffinity column. Proteins in the column eluate were digested with Lys-C endoproteinase, and the resulting peptide fragments were analyzed directly with a highly sensitive LC-MS/MS system (Natsume et al. 2002). The proteins identified by this approach included all subunits of the 26S proteasome with the exception of S5a (Rpn10), S14 (Rpn12), S5b, ß1 and ß7 (Table 1). We thus investigated further the molecular relation between FKBP38 and the 26S proteasome.


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  		Table 1  Proteins identified by LC-MS/MS analysis in immunoprecipitates prepared with anti-FLAG from HEK293T cells expressing 3 x FLAG-tagged FKBP38
 
To confirm the interaction between FKBP38 and the 26S proteasome, we performed a co-immunoprecipitation assay. Immunoprecipitates were prepared with anti-FLAG from the lysates of HEK293T cells transfected with an expression vector for FLAG-tagged FKBP38 (or with the empty vector). The resulting precipitates were then subjected to immunoblot analysis with antibodies to the S2 subunit of the 19S proteasome and with anti-FLAG (Fig. 1A). The S2 subunit was detected in the immunoprecipitates prepared from the cells expressing FLAG-FKBP38. We also examined whether FKBP52, which shares a significant homology with FKBP38 but is localized in the cytosol, and Cyclophilin 40 interacted with the proteasome. FKBP52 weakly bound to the proteasome, whereas Cyclophilin 40 did not (Supplementary Fig. S1). Similar analysis was performed to detect the interaction between endogenous proteins. Immunoprecipitates prepared with anti-FKBP38 or with control IgG were subjected to immunoblot analysis with antibodies to S2, to the {alpha}1 subunit of the 20S proteasome, and to FKBP38 (Fig. 1B, data not shown). Endogenous {alpha}1 and S2 subunits were co-precipitated with endogenous FKBP38, whereas no signal was detected in control immunoprecipitates. These results suggest that endogenous FKBP38 interacts with the 26S proteasome.


Figure 1
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  		Figure 1  Interaction between FKBP38 and the proteasome. (A) HEK293T cells were transiently transfected with an expression vector for 3 x FLAG-tagged FKBP38. Cell lysates were subjected to immunoprecipitation (IP) with anti-FLAG, and the resulting precipitates were subjected to immunoblot analysis (IB) with anti-S2 and anti-FLAG. A portion (5% of the input for immunoprecipitation) of the cell lysates was also subjected directly to immunoblot analysis. (B) Lysates of (non-transfected) HEK293T cells were subjected to immunoprecipitation with anti-FKBP38 (FK38N1) or with control rabbit IgG. The resulting precipitates, as well as a portion (5% of the input for immunoprecipitation) of the cell lysates, were subjected to immunoblot analysis with anti-{alpha}1 and anti-FKBP38.

 
To determine the region of FKBP38 that is responsible for binding to the proteasome, we expressed HA-tagged full-length (FL) FKBP38 or a series of deletion mutants thereof in HEK293T cells (Fig. 2A). Cell lysates were subjected to immunoprecipitation with anti-HA and the resulting precipitates were subjected to immunoblot analysis with anti-S2 and anti-HA (Fig. 2B,C). Whereas FL, {Delta}N, {Delta}N{Delta}FKBP and {Delta}N{Delta}CaM forms of FKBP38 interacted with the proteasome, the {Delta}N{Delta}TPR and {Delta}TPR mutants did not, suggesting that the TPR domain of FKBP38 is necessary for its interaction with the proteasome. A mutant containing only the TPR domain was found to interact with the proteasome, indicating that the TPR domain of FKBP38 is also sufficient for binding to the proteasome.


Figure 2
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  		Figure 2  Identification of the region of FKBP38 that is necessary and sufficient for binding to the proteasome. (A) Schematic representation of the domain organization of full-length (FL) human FKBP38 and a series of mutants thereof. The binding results in (B) and (C) are summarized at the right. CaM, calmodulin-binding domain; TM, transmembrane domain. (B, C) HEK293T cells were transiently transfected with expression vectors for HA-tagged forms of the FKBP38 mutants shown in (A) or with the empty vector (Mock). Cell lysates were then subjected to immunoprecipitation with anti-HA, and the resulting precipitates were subjected to immunoblot analysis with anti-S2 and anti-HA. A portion (5% of the input for immunoprecipitation) of the cell lysates was also subjected directly to immunoblot analysis.

 
To identify the subunit (or subunits) of the proteasome that mediates the interaction with FKBP38, we fractionated the holo-proteasome by SDS-PAGE, transferred the separated proteins to a filter, and incubated the filter with a recombinant His6-tagged form of FKBP38 that lacks the NH2-terminal and transmembrane domains. Immunodetection with anti-His6 suggested that FKBP38 interacted with the S4 subunit of the 19S proteasome (Fig. 3A). To confirm this result, we performed the same assay (Fig. 3B) as well as a "pull-down" assay (Fig. 3C) with recombinant S3, S4 and S5a subunits of the proteasome. Both assays revealed that FKBP38 directly bound to the S4 subunit of the proteasome.


Figure 3
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  		Figure 3  Identification of the proteasome subunit that mediates interaction with FKBP38. (A) The purified holo-proteasome was fractionated by SDS-PAGE, and the separated proteins were transferred to a polyvinylidene difluoride membrane and probed with recombinant His6-tagged FKBP38 (58–393). Complexes were detected with anti-His6 and secondary antibodies (2nd Ab), as indicated. The membrane was also stained with Coomassie brilliant blue (CBB). The positions of protein standards (in kilodaltons) are shown on the left and those of several proteasome subunits are indicated on the right. (B) Recombinant S3, S4 or S5a subunits of the 19S proteasome were expressed as GST-tagged proteins in Escherichia coli and purified with glutathione-conjugated beads, after which the GST tag was proteolytically removed. The interaction of these recombinant proteins (or the 26S proteasome) with recombinant His6-tagged FKBP38 (58–393) was then examined as in (A). The asterisk indicates a non-specific band. (C) GST-tagged recombinant S3, S4 or S5a prepared as in (B) was incubated with His6-FKBP38 (58–393) in vitro, and the GST fusion proteins were then precipitated with glutathione-conjugated beads. The resulting precipitates, as well as a portion (5% or 10%) of the original binding mixtures, were then subjected to immunoblot analysis with anti-His6 and anti-GST.

 
We next investigated the region of the S4 subunit that is responsible for binding to FKBP38. The S4 subunit contains a coiled-coil domain (residues 82–102) and a AAA (ATPase associated with various cellular activities) domain (residues 218–357) (Gorbea et al. 2000). We produced recombinant GST-tagged S4 and three deletion mutants thereof in bacteria (Fig. 4A); the mutants lacked the COOH-terminal domain ({Delta}C1, residues 1–360), the AAA domain ({Delta}C2, residues 1–167), or the coiled-coil domain ({Delta}N, residues 168–440). A pull-down assay revealed that the FL protein, {Delta}C1 and {Delta}C2 bound to His6-FKBP38, whereas the {Delta}N mutant did not (Fig. 4B). These data suggest that the NH2-terminal region of S4 containing the coiled-coil domain is responsible for binding to FKBP38.


Figure 4
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  		Figure 4  Identification of the region of the S4 subunit that is necessary for binding to FKBP38. (A) Schematic representation of the domain organization of the human S4 subunit and a series of mutants thereof. The binding results in (B) are summarized on the right. CC, coiled-coil domain. (B) His6-FKBP38 (58–393) was incubated with GST-tagged forms of the S4 mutants shown in (A) or with GST, and the binding mixtures were then subjected to precipitation with glutathione-conjugated beads. The resulting precipitates, as well as a portion (5% or 30%) of the original binding mixtures, were subjected to immunoblot analysis with anti-His6 and anti-GST.

 
The 26S proteasome is localized to mitochondria

We previously showed that FKBP38 is localized mainly to the mitochondrial outer membrane (Shirane & Nakayama 2003). We thus examined whether the 26S proteasome also localizes to mitochondria. COS-7 (African green monkey kidney), HeLa (human cervical cancer) or NIH 3T3 (mouse fibroblast) cell lines were subjected to immunofluorescence staining with antibodies to the S4 (Fig. 5) or {alpha}3 (data not shown) subunits of the proteasome in conjunction with staining of mitochondria with MitoTracker. In all three cell lines, both endogenous S4 (19S proteasome) and {alpha}3 (20S proteasome) subunits were found to localize in part to mitochondria. Together, these data suggest that FKBP38 and the 26S proteasome interact in vivo at the mitochondrial surface.


Figure 5
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  		Figure 5  Mitochondrial localization of the proteasome. COS-7 (A), HeLa (B), or NIH 3T3 (C) cells were incubated with MitoTracker (for staining of mitochondria) for 20 min, fixed and immunostained with anti-S4. Individual and merged images are shown, with the regions of the merged images delineated by white boxes being enlarged in the rightmost panels.

 
FKBP38 functions to tether the 26S proteasome to the organellar membrane

To examine the relation of FKBP38 to proteasome function, we compared the abundance and activity of the proteasome between lysates of MEFs derived from Fkbp38+/+ and Fkbp38–/– mice. We prepared cytosolic and membrane fractions of the cell lysates, with mitochondria being enriched in the latter fraction as revealed by the presence of the mitochondrial membrane protein TOM70 and the absence of the cytosolic protein {alpha}-tubulin (Fig. 6). The abundance of S2 (19S) and {alpha}6 (20S) subunits of the proteasome was markedly reduced in the membrane fraction of Fkbp38–/– MEFs compared with that in the corresponding fraction of Fkbp38+/+ MEFs. Immunofluorescence staining revealed that the mitochondrial targeting of the 26S proteasome was relatively decreased in Fkbp38–/– MEFs compared with that in Fkbp38+/+ MEFs (Supplementary Fig. S2). These data thus suggest that FKBP38 functions to tether the 26S proteasome to the organellar membrane.


Figure 6
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  		Figure 6  Proteasome abundance in subcellular fractions of Fkbp38+/+ and Fkbp38–/– MEFs. MEF lysates or cytosolic or membrane fractions derived therefrom (10 µg of protein) were subjected to immunoblot analysis with antibodies to the indicated proteins.

 
We finally examined the activity of the proteasome in the cytosolic and membrane fractions of Fkbp38+/+ and Fkbp38–/– MEF lysates. There was no significant difference in proteasome activity in the whole lysate (Fig. 7A) or in the cytosolic fraction (Fig. 7B) between Fkbp38+/+ and Fkbp38–/– MEFs. Given that the activity was almost completely abolished by the proteasome inhibitor MG132, the assay indeed appeared to reflect the activity of the proteasome. The proteasome activity in the membrane fraction of Fkbp38–/– MEFs was significantly reduced compared with that in the corresponding fraction of Fkbp38+/+ MEFs (Fig. 7C). Together with the data in Fig. 6, these results thus indicate that FKBP38 plays a key role in anchoring the proteasome at the organellar membrane.


Figure 7
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  		Figure 7  Proteasome activity in subcellular fractions of Fkbp38+/+ and Fkbp38–/– MEFs. MEF lysates (A) or cytosolic (B) or membrane (C) fractions thereof were incubated at 37 °C with the substrate Suc-LLVY-AMC in the absence or presence of MG132. The accumulation of 7-amino-4-methyl coumarin (AMC) was assayed fluorometrically and expressed as nanomoles per minute per milligram of protein. Data are means ± SEM of values from three independent experiments. *P < 0.05 (Student's t test). The P values are 0.0572, 0.1281 and 0.0006 in (A–C), respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
The intracellular distribution of the proteasome, especially in yeast and mammalian cells, has been examined in several previous studies (Rivett 1998; Hirsch & Ploegh 2000; Wojcik & DeMartino 2003). Immunolabeling, tagging with green fluorescent protein, immuno-electron microscopy and subcellular fractionation methods have thus shown that the proteasome is present in the cytoplasm and in the nucleus. In the cytoplasm, it is associated with various compartments, including the centrosome, ER, cis-Golgi and endosomes, as well as with the cytoskeleton, but the anchors that link the proteasome to these structures have remained unknown. Recently, several candidates for such anchoring molecules have been proposed, including Ecm29 for the centrosome, ER and endosomes (in mammalian cells) (Gorbea et al. 2004), translocon for the ER (in budding yeast) (Kalies et al. 2005) and Cut8 for the nucleus (in fission yeast) (Takeda & Yanagida 2005). We have now presented several lines of evidence that FKBP38, a chaperone that resides in the mitochondrial outer membrane and ER membrane, anchors the 26S proteasome to such organelles. Immunostaining thus revealed that a subset, but not all, of the proteasome complexes in COS-7, HeLa or NIH 3T3 cells co-localized with mitochondria. Furthermore, cell fractionation revealed a substantial difference in the abundance and activity of the proteasome in the membrane fraction between wild-type and FKBP38-deficient MEFs. It is likely that this difference is the result of a defect in anchoring of the proteasome to mitochondria by FKBP38 in the latter cells. Together, these data indicate that FKBP38 serves to anchor the proteasome to organellar membrane.

We found that FKBP38 binds directly via its TPR domain to the S4 subunit of the 19S proteasome. Given that Rpt2, a yeast orthologue of the mammalian S4 proteasome subunit, is considered to be responsible for gating of the 20S proteasome (Rubin et al. 1998; Kohler et al. 2001), it is possible that FKBP38 regulates not only the localization of the proteasome but also, through an effect on the gating state of the 20S proteasome, its activity.

FKBP38 has multiple binding partners, including Bcl-2 and Bcl-xL (Shirane & Nakayama 2003), calcineurin (Shirane & Nakayama 2003), protrudin (Shirane & Nakayama 2006) and the proteasome (this study). It is unclear whether interactions between FKBP38 and the proteasome and its other binding partners are independent or linked biologically. The regions of FKBP38 that are required for interaction with the proteasome or with Bcl-2 or Bcl-xL appear to differ. Binding to the S4 subunit of the proteasome is thus solely dependent on the TPR domain of FKBP38, whereas broader regions of the protein are required for the association with Bcl-2 or Bcl-xL. The existence of multiple binding partners of molecular chaperones is not unusual. For example, FKBP12, a founding member of the FKBP family, interacts with various proteins including ryanodine receptor 1 (Brillantes et al. 1994), the inositol 1,4,5-trisphosphate receptor (Cameron et al. 1995), the type 1 receptor for transforming growth factor-ß (Wang et al. 1996), TRPC channels (Sinkins et al. 2004), YY1 (Yang et al. 1995), FAP48 (Chambraud et al. 1996) and amyloid precursor protein (Liu et al. 2006). FKBP38 contains multiple domains, including three TPRs, a calmodulin-binding domain and a transmembrane domain in addition to its FKBP domain, whereas FKBP12 consists almost exclusively of its FKBP domain. Given the larger size and more complex structural organization of FKBP38 compared with those of FKBP12, it is likely that FKBP38 indeed interacts with multiple partners in vivo.

In yeast, mitochondria constantly release peptides (most comprising 8–15 amino acids) derived from the degradation of non-assembled mitochondrial proteins, many of which reside in the inner membrane and matrix (Augustin et al. 2005). One such degradation product has been detected at the surface of mammalian cells in association with major histocompatibility complex class I molecules (Loveland et al. 1990). Furthermore, the E2 component of the 2-oxoglutarate dehydrogenase complex, a rate-limiting enzyme of the Krebs cycle in the mitochondrial matrix, was shown to be ubiquitylated and degraded by the proteasome (Habelhah et al. 2004). More recently, 5-aminolevulinate synthase 2, which catalyzes the first and rate-limiting step of heme synthesis within the mitochondrial matrix, was also found to undergo ubiquitin-dependent proteolysis by the proteasome (Abu-Farha et al. 2005). FKBP38-mediated tethering of the proteasome to mitochondria may thus play an important role in these processes.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Protein identification by LC-MS/MS analysis

FKBP38-associated proteins were digested with Achromobacter protease I, and the resulting peptides were analyzed with a nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS) system as described previously (Natsume et al. 2002; Komatsu et al. 2004). The peptide mixture was applied to a Mightysil-PR-18 (particle size, 1 µm; Kanto Chemical) fritless column [45 mm by 0.150 mm (inner diameter)] and fractionated over 30 min at a flow rate of 50 nL/min with a 0%–40% gradient of acetonitrile in 0.1% formic acid. Eluted peptides were sprayed directly into a quadropole time-of-flight hybrid mass spectrometer (Q-Tof Ultima; Micromass, Manchester, UK). MS and MS/MS spectra were obtained in a data-dependent mode. Up to four precursor ions above an intensity threshold of 10 counts/s were selected for MS/MS analyses from each survey scan. All MS/MS spectra were searched against protein sequences of Swiss Prot and RefSeq (NCBI) with the use of batch processes of the MASCOT software package (Matrix Science, London, UK). The criteria for match acceptance included: (i) If the match score exceeded the threshold by 10, identification was accepted without further consideration. (ii) If the difference between the score and threshold was < 10, or if a protein was identified on the basis of a single matched MS/MS spectrum, we manually confirmed the raw data before acceptance. (iii) Peptides assigned by fewer than three y series ions and peptides with a charge of +4 were eliminated regardless of their scores.

Construction of expression vectors

Construction of vectors encoding human FKBP38 and its deletion mutants was described previously (Shirane & Nakayama 2003). Complementary DNAs encoding the human proteasome subunits S3 and S4 were kindly provided by K. Tanaka (Tokyo Metropolitan Institute of Medical Science) (Kominami et al. 1997), whereas that encoding the human proteasome subunit S5a was amplified by the polymerase chain reaction from HeLa cell cDNAs with the primers 5'-ATAGAATTCCATGGTGTTGTTGGAAAGCACTATGGT-3' and 5'-ATACGCGTCGACTCACTTCTTGTCTTCCTCCTTCTT-3'. These cDNAs were subcloned into the pGEX-6p vector (Amersham Biosciences, Piscataway, NJ).

Cell culture and transfection

HEK293T cells, HeLa cells, COS-7 cells, NIH 3T3 cells and mouse embryonic fibroblasts (MEFs) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). HEK293T cells were transfected with expression vectors with the use of the FuGene 6 reagent (Roche Applied Science, Indianapolis, IN). MEFs were isolated from Fkbp38–/– mice (M.S. and K.I.N., manuscript in preparation) as described previously (Nakayama et al. 1996, 2000).

Antibodies

Rabbit polyclonal antibodies (FK38N1) to FKBP38 were produced as described previously (Shirane & Nakayama 2003). Rabbit polyclonal antibodies to TOM70 were kindly provided by K. Mihara (Kyushu University) (Suzuki et al. 2000). A mouse monoclonal antibody (IB5) to the {alpha}1 subunit and rabbit polyclonal antibody to the {alpha}6 subunits of the 20S proteasome were obtained from ICN/Chappel (Aurora, OH) and Abcam (Cambridge, UK), respectively; rabbit polyclonal antibodies to the S2 or S4 subunits of the 19S proteasome were from Abcam and Biomol (Plymouth Meeting, PA), respectively; a mouse monoclonal antibody to glutathione S-transferase (GST) was from MBL (Nagoya, Japan); rabbit polyclonal antibodies (H-15) to hexahistidine (His6) were from Santa Cruz Biotechnology (Santa Cruz, CA); a mouse monoclonal antibody (M5) to the FLAG epitope was from Sigma (St. Louis, MO); a mouse monoclonal antibody (HA11) to the hemagglutinin epitope (HA) was from Research Diagnostics (Flanders, NJ); and a mouse monoclonal antibody (TU01) to {alpha}-tubulin was from Zymed (San Francisco, CA); and rabbit polyclonal antibodies to calnexin were from Stressgen (Victoria, Canada).

Immunoprecipitation and immunoblot analysis

Cells were lysed in a solution containing 40 mM HEPES–NaOH (pH 7.6), 150 mM NaCl, 10% glycerol, 0.5% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride. The lysates were then incubated for 20 min at 4 °C with antibodies to FLAG (1 µg/mL), to HA (1 µg/mL) or to FKBP38 (4 µg/mL) and with protein G-Sepharose beads (Amersham Biosciences). The resulting immunoprecipitates were washed 3 times with a solution containing 40 mM HEPES–NaOH (pH 7.6), 150 mM NaCl, 5 mM EDTA, 10% glycerol and 0.1% Triton X-100. Immunoblot analysis was performed as described (Kamura et al. 2004).

Blot analysis of protein interaction

The human holo-proteasome (2 µg; Immatics, Tübingen, Germany) or recombinant human subunits of the proteasome were resolved by SDS–polyacrylamide gel electrophoresis (PAGE) on a 10% gel, and the proteins were transferred to a polyvinylidene difluoride membrane. The membrane was incubated at room temperature first for 90 min with 3% non-fat milk in Tris-buffered saline (TBS) and then for 60 min with the His6-tagged FKBP38-{Delta}TM mutant (amino acids 58–393) at a concentration of 0.1 mg/mL in TBS. After washing twice with TBS for 10 min, the membrane was incubated for 1 h at room temperature with anti-His6. The membrane was again washed twice with TBS for 10 min, after which immune complexes were detected with horseradish peroxidase-conjugated antibodies to rabbit immunoglobulin G (IgG) (Promega, Madison, WI) and enhanced chemiluminescence reagents (Amersham Biosciences).

Immunofluorescence staining

COS-7, HeLa or NIH 3T3 cells grown on glass coverslips were fixed for 10 min at –20 °C with methanol and then incubated for 1 h at room temperature with antibodies to the S4 subunit of the 19S proteasome in phosphate-buffered saline containing 0.5% bovine serum albumin and 0.1% saponin. Immune complexes were detected with Alexa 488-labeled goat polyclonal antibodies to rabbit IgG (Molecular Probes, Eugene, OR) at a dilution of 1 : 2000. For staining of mitochondria, cells were incubated for 20 min with 100 nM MitoTracker (Molecular Probes) before fixation. The cells were finally covered with a drop of Gel/Mount (Biomeda, Foster City, CA) and examined with a confocal fluorescence microscope (Radians 2000, BioRad Laboratories, Hercules, CA).

In vitro binding assay

Recombinant GST- or His6-tagged proteins were prepared in bacteria, and an in vitro binding assay was performed as described previously (Takahashi et al. 2005).

Assay of proteasome activity

Proteasome activity was assayed as described previously (Canu et al. 2000) with some modifications. In brief, MEFs were lysed in a solution containing 20 mM Tris–HCl (pH 7.2), 150 mM NaCl, 0.1 mM EDTA, 1 mM 2-mercaptoethanol, 5 mM ATP, 20% glycerol and 0.1% Triton X-100. The lysate was centrifuged at 20 000 g for 20 min at 4 °C, the resulting supernatant was saved as the cytosolic fraction, and the resulting pellet was further lysed in the same lysis buffer with the exception that the concentration of Triton X-100 was 0.5%. The treated pellet was centrifuged at 20 000 g for 20 min at 4 °C, and the resulting supernatant was saved as the membrane fraction. The cytosolic and membrane fractions as well as MEF lysates prepared with a lysis solution containing 0.5% Triton X-100 were incubated at 37 °C with 50 µM Suc-LLVY-AMC (BostonBiochem, Cambridge, MA) in the absence or presence of 1 µM MG132 (Peptide Institute, Osaka, Japan). Hydrolysis of the peptide substrate was measured at 0, 30 and 60 min with excitation and emission wavelengths of 380 and 460 nm.


   Acknowledgements
 
We thank K. Tanaka for the S3 and S4 cDNAs; K. Mihara for antibodies to TOM70; R. Mitsuyasu, N. Nishimura and other laboratory members for technical assistance; and A. Ohta and M. Kimura for help in preparation of the manuscript.


   Footnotes
 
Communicated by: Noriko Osumi

* Correspondence: E-mail: nakayak1{at}bioreg.kyushu-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Abu-Farha, M., Niles, J. & Willmore, W.G. (2005) Erythroid-specific 5-aminolevulinate synthase protein is stabilized by low oxygen and proteasomal inhibition. Biochem. Cell Biol. 83, 620–630.[CrossRef][Medline]

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Accepted: 20 February 2007




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