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Abstract. Hereditary spastic paraplegia describes a diverse group of disorders characterized by progressive paraparesis primarily affecting lower limbs. In Troyer ...

Journal of Neurochemistry, 2006, 98, 1908–1919

doi:10.1111/j.1471-4159.2006.04008.x

The hereditary spastic paraplegia protein spartin localises to mitochondria JianPing Lu, Faiza Rashid and Paula C. Byrne UCD School of Medicine and Medical Science, Conway Institute, University College Dublin, Ireland

Abstract Hereditary spastic paraplegia describes a diverse group of disorders characterized by progressive paraparesis primarily affecting lower limbs. In Troyer syndrome, an autosomal recessive form of hereditary spastic paraplegia, patients have dysarthria, distal amyotrophy, developmental delay and short stature in addition to spastic paraparesis. It is caused by a frameshift mutation (1110delA) in SPG20 leading to premature truncation of spartin, a protein with no known function. The objective of this study was to determine the subcellular localization of spartin and investigate the effect of the 1110delA mutation. We observed cytoplasmic expression of spartin in all transfected cell lines. Using superimposed organelle markers or immunocytochemistry staining, we established that spartin localizes to mitochondria and that this localization is dependent on sequences in

the C-terminal region. Mutant spartin containing the 1110delA mutation has lost mitochondrial localization. Immunocytochemistry staining using anti-alpha-tubulin antibody provided evidence for partial co-localization of spartin with microtubules. Analysis of fluorescence resonance energy transfer indicated that sequences in the amino terminal are important in mediating microtubule interaction. This study provides the first evidence of spartin subcellular localization and identifies it as the third mitochondrial protein implicated in hereditary spastic paraplegia. Our results suggest that Troyer syndrome may be due to defective microtubulemediated trafficking of mitochondria and/or mitochondrial dysfunction. Keywords: fluorescence resonance energy transfer, hereditary spastic paraplegia, mitochondria, spartin, SPG20. J. Neurochem. (2006) 98, 1908–1919.

Hereditary spastic paraplegia (HSP) describes a genetically and clinically diverse group of inherited neurodegenerative disorders that are characterized by progressive spastic paraparesis, primarily affecting the lower limbs (Fink 2002). To date, 28 HSP causing loci have been mapped and designated SPG1 to SPG29 in order of discovery (SPG22 has been reserved but no data have yet been published). Three of these loci are X-linked (SPG1, SPG2 and SPG16), 12 are autosomal dominant (SPG3, SPG4, SPG6, SPG8, SPG8, SPG10, SPG12, SPG13, SPG17, SPG18, SPG19 and SPG29) and 13 are autosomal recessive (SPG5, SPG7, SPG11, SPG14, SPG15, SPG20, SPG21, SPG23, SPG24, SPG25, SPG26, SPG27 and SPG28) [access the HUGO Gene Nomenclature Committee (http://www. gene.ucl.ac.uk/nomenclature) for description]. Pathologically, HSP is characterized by degeneration of the longest axons in the body, the corticospinal tracts and, to a lesser extent, the fasiculi gravis. HSP has traditionally been classified into ‘pure’ HSP, when spastic paraparesis is the only symptom, and ‘complicated’ HSP, where affected patients suffer additional neurological and non-neurological symptoms.

Troyer syndrome is a ‘complicated’ form of autosomal recessive (AR) HSP with a high incidence in the Old Order Amish, where affected patients have dysarthria, distal amyotrophy, mild developmental delay and short stature in addition to progressive spastic paraparesis. A mutation in SPG20 on chromosome 13q has been identified in a family with Troyer syndrome (Patel et al. 2002). SPG20 encodes spartin, a protein with 666 amino acid residues that is ubiquitously expressed both within the nervous system and in non-neuronal tissues. The effect of the identified frame-

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Received January 25, 2006; revised manuscript received April 20, 2006; accepted May 17, 2006. Address correspondence and reprint requests to Paula C Byrne, UCD School of Medicine and Medical Sciences, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland. E-mail: [email protected] Abbreviations used: AD, autosomal dominant; ATRA, all-trans retinoic acid; DAPI, 4¢,6-diamidino-2-phenylindole; ECFP, enhanced cyan fluorescent protein; EEA, early endosome antigen; EGFP, enhanced green fluorescent protein; FRET, fluorescence resonance energy transfer; HSP, hereditary spastic paraplegia; MIT, microtubule interacting and trafficking; PBS, phosphate-buffered saline.

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shift mutation in exon 4 is a predicted substitution of 29 amino acids after the mutation and a protein truncated by 268 residues. The function of spartin is unknown. Bioinformatic analysis found strong sequence similarity between the C-terminal region of spartin and a number of uncharacterized plant proteins associated with senescence. More informative is the presence of a microtubule interacting and trafficking (MIT) domain in the amino terminal region of the protein. This MIT domain is found in a number of proteins including the endosomal protein, VPS4, that has a well defined role in intracellular protein trafficking (Ciccarelli et al. 2003). The MIT domain is also present in spastin, a protein which, when mutated, causes the most common form of HSP (Hazan et al. 1999). In spastin, the MIT domain is required to mediate spastin interaction with Chromatin modifying protein 1B (CHMP1B), a protein associated with ESCRT-III (endosomal sorting complex required for transport) (Reid et al. 2005). Spastin is a multi-functional protein that also has a role to play in mediating microtubule dynamics (Errico et al. 2002). It was recently shown that spastin is capable of severing microtubules in vitro, while HSPassociated mutations that abolish the ATPase activity of spastin have also lost severing capability (Evans et al. 2005). Immunofluorescent studies show a concentration of spastin in cell regions associated with an active microtubule network: the centrosome at cell division, and growth cones and branching regions in post-mitotic neurones (Errico et al. 2004). Spastin is also found in the endoplasmic reticulum, and localization to the endoplasmic reticulum is dependent on Nterminal sequences that include the MIT domain (unpublished data). The exact pathogenesis of spastin-associated HSP remains unclear, but it seems that defective cellular transport mechanisms due to impaired microtubule networks and/or intracellular membrane trafficking systems may be responsible. Defects identified in two mitochondrial proteins, paraplegin (SPG7) and HSP60 (SPG13), suggest that mitochondrial dysfunction also has a role to play in the pathogenesis of HSP (Casari et al. 1998; Hansen et al. 2002). The occurrence of the MIT domain in spartin suggests that spartin may also be involved in intracellular trafficking. Recently, cell fractionation studies indicated that spartin was primarily a cytoplasmic protein, with a small portion associated with membranes (Bakowska et al. 2005). A yeast two hybrid screen using the amino terminal region of spartin, which encompasses the MIT domain as bait, identified spartin interaction with Eps15, a protein involved in receptormediated endocytosis of epidermal growth factor (Bakowska et al. 2005). No other studies regarding subcellular localization or function of spartin have been reported. In an attempt to clarify the subcellular localization of spartin and to gain insight into its physiological function, we performed expression and cell localization experiments using fluorescent-tagged, full-length and mutant spartin together with organelle-specific stains to identify with which cellular

components spartin associated. We have established that spartin is located inside the mitochondria and that this localization is determined by sequences in the C-terminal region. Our results also support the central role that microtubules appear to play in the general pathogenesis of HSP. Using fluorescence resonance energy transfer (FRET) examination, we have shown that spartin associates with the microtubule network in the cytoplasm, and that this association is dependent on sequences in the N-terminal region of the protein. We have created a spartin construct that mimics the mutation causing Troyer syndrome, and we have found that this prematurely truncated spartin has lost its mitochondrial localization but has retained the ability to interact with microtubules. Axonal degeneration in Troyer syndrome may be due to the loss of normal spartin function caused by dislocation of the protein from mitochondria, and/or to interference of normal microtubule function by the presence in the cytoplasm of truncated spartin protein.

Materials and methods Preparation of full-length and mutant spartin cDNA and construction of expression plasmids Full-length spartin cDNA was amplified via a nested PCR approach using normal human adult brain cDNA (GenePool cDNA, Cat no. D8030-01, Invitrogen, Carlsbad, CA, USA) as a template. To facilitate cloning, primer pairs were synthesized to contain restriction enzyme sites (NdeI and HindIII) according to SWISS-PROT human spartin cDNA sequence (SPG20_HUMAN, Q8N0X7; Ensembl Gene ID ENSG00000133104). The expression frame of full-length spartin cDNA was subcloned between the NdeI and HindIII restriction enzyme sites of pDNR (Clontech, Mountain View, CA, USA) and transferred into mammalian expression vector pLP-ECFP-C1 (Clontech) through Cre-LoxP recombinase reaction. The resulting vector (pECFP-SPG20) expresses full-length spartin fused in frame with enhanced cyan fluorescent protein (ECFP) (Fig. 1). A plasmid was also prepared that would express the untagged full-length SPG20 sequence (pSPG20) by digesting pLPCFP-SPG20 with Age I and Sal I to remove the ECFP tag, purifying the relevant DNA fragment (7024 bp) and filling in the ends with Klenow prior to re-ligation. The resulting product was transformed into E. coli JM109 and the plasmid sequenced to confirm that the desired product had been obtained. To examine the role played by the amino and carboxyl regions of spartin in determining cell localization, we created prematurely truncated spartin and amino terminal deleted spartin fused in frame with ECFP. The prematurely truncated spartin, consisting of amino acids 1–347 fused to ECFP, was created by digesting pECFP-SPG20 with EcoRI and MunI and re-ligating the purified vector to generate pECFP-SPG201)347. Amino terminal deleted spartin was created by digesting pECFP-SPG20 vector with AccI and EcoRI, treating with Klenow to fill in the ends and re-ligating to produce pECFPSPG20347)666, which would express the carboxyl region of the protein tagged with ECFP. Using site directed mutagenesis and a series of amplification and cloning steps, we incorporated the frameshift deletion (1110delA)

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ECFP- SPG201110delA

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ECFP-SPG20347-666

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Fig. 1 Schematic representation of normal and mutant ECFP-spartin fusion proteins.

29 substituted amino acids

that causes Troyer syndrome. pECFP-SPG20 was used as a template to amplify two overlapping fragments spanning the site of the mutation. The 1110delA mutation was incorporated into the first fragment using a reverse primer that included the appropriate sequence change. To facilitate cloning, an additional nucleotide change was incorporated that resulted in the generation of a MunI restriction site immediately downstream of the frameshift deletion. The forward primer in this amplification reaction contained the recognition sequence for XmaI that is present in spartin. The second overlapping PCR fragment was amplified using a forward primer that incorporated a MunI restriction site to allow both fragments to be ligated; in this case the reverse primer was complementary to the vector sequence containing a BcuI/SpeI recognition site. PCR products were digested with MunI prior to purification and ligation. This new fragment of DNA was then digested with XmaI and BcuI, then cloned into pECFP-SPG20 that had also been digested with XmaI and BcuI. The resulting clones were sequenced to confirm that only the desired changes had been incorporated. The resulting vector, designated pECFP-SPG201110delA, expresses a prematurely truncated spartin that mimics the mutant spartin found in Troyer syndrome (fs369–398x399) (Ciccarelli et al. 2003). A schematic representation of the proteins expressed by these vectors is shown in Fig. 1. Cell culture and transfection Lipofectamine 2000 (11668–027; Invitrogen) was used for all cell transfections. The cells were transfected, at 50–90% confluence, using 1.0–1.5 lg DNA and 2–3 lL lipofectamine in 2 mL OptiMEM (Invitrogen) per well for six-well plates. After 3–4 h of transfection, the transfection medium was replaced with culture medium. In some experiments, the replacement culture medium was composed of reduced fetal calf serum (FCS) and ionomycin or alltrans retinoicacid (ATRA) to induce differentiation. Geneticin (G418 Sigma, A1720; Sigma-Aldrich, St Louis, MO, USA) was supplemented in sustained culture for selective continuous incubation of cells expressing the desired fusion protein. Where necessary, microtubules were stabilized with Taxol (B5683; Sigma-Aldrich). Immunocytochemistry, organelle tracker staining and confocal microscopy At 1–4 days after transfection, the cells were rinsed with phosphatebuffered saline (PBS) and fixed with 4% paraformaldehyde in 1·

PBS solution. Cells were permeabilized with 0.3%Triton X-100 in 1· PBS for 20 min and washed in PBS. Non-specific reactions were blocked with 5% bovine serum albumin and 5% goat serum in PBS for 30 min before the addition of antibodies. Cells were incubated with labelled antibody or, first and labelled secondary antibody sequentially and washed with PBS. The antibodies used are listed in Table 1. The anti-spartin antibody was produced against synthetic peptides representing amino acids 32–46 and amino acids 311–325. These regions have 100% homology between human and mouse. Rabbits were immunized with peptides and the reaction of the serum tested by ELISA. The serum was affinity purified for immunocytochemistry. Nuclei were stained with 4¢,6-diamidino-2-phenylindole (DAPI; Sigma). The cells were post-fixed with 2% paraformaldehyde in 1· PBS solution, mounted in VectaShield H1000 (Vector Laboratories, Burlingame, CA, USA) and stored at 4C. Organelle tracker staining was undertaken according to the manufacturer’s protocol (Molecular Probes, Invitrogen, Eugene, OR, USA). Cells stained with ER-Tracker Blue-White DPX and MitoTracker Red CMXRos (Molecular Probes, Invitrogen) were photographed following treatment with 4% paraformaldehyde in 1· PBS solution or acetone fixation, respectively. Cells stained with LysoTracker Red DND-99 (Molecular Probes, Invitrogen) were observed in the culture medium directly after staining with no further manipulation. All transfection experiments, organelle tracker staining and immunocytochemistry were performed at least three times, each on different occasions. For each experiment, the results were observed with a Zeiss fluorescent microscope and the results recorded using AXIOVISION software (Zeiss, Go¨ttingen, Germany). The same cell preparations were scanned with a Meta 510 confocal microscope (Zeiss). Cells from the four corners and one central part of the coverslip were squared before scanning, and one or more fields from each square were scanned to reduce subjective selection of individual cells of interest. The scanning stack size was set at 1024 · 1024 (occasionally at 2048 · 2048) pixels. The objective lens used in the confocal image scanning was 63· oil Plan Apochromat (Zeiss), pixel size 0.11 lm. The offset of the threshold was set to collect the weakest to strongest signal with minimum background noise. The pinhole was set near 1.0, with slice thickness set to 0.5 or 1.0 lm for different tracks for DAPI, Rhodamine and FITC. For comparison of the intensity of enhanced green fluorescent

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Table 1 Antibodies and cell organelle-labelling reagents used in immunofluorescence studies

Primary antibodies Mitochondriaa EEA1b Calreticulin Alpha-tubulin Neurofilament GFAP Vimentin Secondary antibodies Goat anti-mouse IgG Goat anti-rabbit IgG Goat anti-rabbit IgG Other labelling reagents Phalloidin ER-Tracker Blue-White DPX MitoTracker Red CMXRos LysoTracker Red DND-99

Type

Label

Source

Monoclonal Monoclonal Rabbit anti-serum Monoclonal Monoclonal Monoclonal Monoclonal

No No No No No Cy3 Cy3

Abcamc (ab14730) Abcam (ab15846) Abcam (ab2907) Molecular Probesd (11126) Sigma (N0142) Sigma (C9205) Sigma (V5255)

Rhodamine Red-X Texas Red HRP

Molecular Probes (R6393) Vector Laboratories (TI-1000) Dakoe (P0448)

Rhodamine

Sigma (P-1951) Molecular Probes (E12353) Molecular Probes (M-7512) Molecular Probes (L-7528)

a

Mitochondria ATP synthase subunit beta. Early endosome antigen 1. c (Cambridge, UK). e (Carpinteria, CA, USA). b

protein (EGFP) and, Rhodamine signal among the groups, the lesser source power outputs were pre-set at 50% for UV laser and 75% for argon laser. The excitation value of the laser was set to 10% at 364 nm, 50% at 458 nm and 80% at 543 nm. DAPI staining was used to show the contour of the nucleus. All confocal images were reviewed with the LSM IMAGE VIEWER software for the Meta 510 Confocal Microscope (Zeiss). Slices through the cell equator, or those showing the best structures, were selected for photographs using the ‘copy and paste’ tool. Adobe Photoshop (Adobe Systems Inc., San Jose, CA, USA) was used only if necessary to re-size photos without any modification. Fluorescence resonance energy transfer (FRET) HeLa cells were double-transfected with pEYFP-Tub (6118–1; BD Biosciences) and one of the vectors expressing ECFP-spartin fusions, pECFP-SPG20, pECFP-SPG201110delA or pECFPSPG20347)666. HeLa cells transfected with pEYFP or pECFP only, or double-transfected with pEYFP and pECFP, and pEYFP-Tub and pECFP-SPG4 (spastin), were used as controls. Twenty consecutive positive cells expressing both EYFP-alpha tubulin and ECFP-spartin, or one of the mutant fusion proteins, were subjected to the following FRET examination. From each cell, one to three regions of interest (ROI) were set for photobleaching. The laser of the Zeiss LSM510 Meta confocal microscope was tuned to lines at 458, 488 and 514 nm. An established filter setting (Karpova et al. 2003) was used to optimize the imaging of ECFP and EYFP, and to eliminate cross-talk between the channels. Photomultiplier (PMT) settings established by the previous procedure consistently yielded no cross-talk when ECFP-only or EYFP-only cells were imaged. FRET was measured using the acceptor photobleaching method (Kenworthy 2001). The FRET energy transfer efficiency was calculated as percentage of (Ef): Ef ¼ (Iafter – Ibefore) · 100/

Iafter, where Ibefore/after is the ECFP intensity before or after the photobleaching. This formula yields the increase in ECFP fluorescence following EYFP bleach normalized by ECFP fluorescence after the bleach. Similar calculations in non-bleached regions of the specimen: Cf ¼ (Iafter – Ibefore) · 100/Iafter were always performed on the same cell as controls (Karpova et al. 2003).

Results

Localization of spartin to the cytoplasm To gain insight into spartin subcellular localization, we examined spartin expression in a variety of neuronal and non-neuronal cells, including neuronal cells from primary culture, human neuroblastoma SH-SY5Y and murine neuroblastoma neuro-2A cell lines, at various stages of differentiation, and non-neuronal rat glioma C6 cells as well as HeLa cells. Spartin expression was monitored using fluorescent confocal microscopy to ECFP-tagged protein. HeLa and SHSY5Y cells transfected with pLP-ECFP displayed a diffuse cytoplasmic distribution, indicating that the ECFP tag does not target any organelle. For ECFP tagged spartin, we observed cytoplasmic expression (Figs 2a–d) in all transfected cells examined. We did not observe any evidence of nuclear expression, even when ECFP-labelled spartin was expressed at high levels (Fig. 2d). In primary cultured neurone cells, the distribution was mostly dotty or punctuate, suggesting association with cellular organelles. This dotty expression could be seen both in the cell body and in the extending neurites. In cultured neuroblastoma cells expres-

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Fig. 2 Cytoplasmic (mitochondrial) expression of full-length spartin in neuronal (primary mouse and rat neurone, and SHSY5Y human neuroblastoma) cells. (a–d) Cytoplasmic expression of spartin in primary mouse (a), rat neurone (b, c) and SH-SY5Y human neuroblastoma cells (d) expressing full-length ECFP-spartin and counterstained with DAPI. The protein may form vesicular structures (c). (e–l) Spartin is localized to mitochondria in primary mouse neurone (e, f), SH-SY5Y (g–j) and HeLa (k, l) cells expressing full-length ECFP-spartin superimposed with MitoTracker (e–g, k) or anti-mitochondria antibody (h–j, l) and counterstained with DAPI. Images were acquired as described with stack size set to 1024 · 1024, with the exception of (j) which had stack size set at 2048 · 2048.

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sing ECFP-tagged spartin, the dotty distribution pattern was observed together with larger aggregations or clusters of dots accumulated at one side of the nucleus (Fig. 2d). A weak diffuse cytoplasmic signal could also be seen, and ECFPlabelled spartin appeared as vesicle-like structures in the cells (Fig. 2c). To confirm that the subcellular expression pattern we observed was not influenced by the presence of the ECFP tag, we developed rabbit polyclonal antibodies against different regions of spartin (amino acids 32–46 and 311– 325). Immunocytochemistry showed that the immunoreaction completely co-localized with the spartin-ECFP fusion protein, and that the reaction was completely blocked by pre-incubation with the above-synthesized peptides. Immunofluorescence studies of HeLa cells transfected with full-length untagged spartin (pSPG20), followed by immunoreaction with anti-spartin antibody, confirmed the localization of spartin in mitochondria and that the observed distribution of ECFP-tagged spartin was not influenced by the presence of the ECFP tag (data not shown). The mitochondria are the major site of spartin localization The punctuate cytoplasmic expression pattern observed for spartin is suggestive of localization (packaging and/or functioning) inside cellular vesicles or organelles. To investigate this, we used a series of organelle-specific markers (trackers) and antibodies to show the relationship between these organelles and full-length ECFP-labelled spartin. Following transfection of primary cultured mouse neurones, neuronal SH-SY5Y and non-neuronal HeLa cells with pECFP-SPG20, the cells were exposed either to organelle-specific trackers for endoplasmic reticulum, mitochondria or lysosomes, or to one of the following organellespecific antibodies: anti-mitochondria ATP synthase subunit beta for mitochondria, anti-calreticulin to visualize endoplasmic reticulum, or anti-early endosome antigen 1 (EEA1) antibody to show early endosomes. As can be seen from Figs 2(e–l), ECFP-spartin co-localizes with mitochondria in mouse neurone, human neuroblastoma (SH-SY5Y) and HeLa cells. Spartin co-localization with mitochondria was also observed in rat glioma C6 cells and mouse neuroblastoma neuro2A cells. This result was consistently observed using the widely accepted mitochondrialspecific marker, MitoTracker. Mitochondrial localization was further confirmed by immunocytochemistry with antibody to mitochondrial ATP synthase subunit beta (another mitochondria marker). This is the first reported observation of the localization of spartin in cell organelles. Using confocal microscopy, we were able to optimize both the intensity of signal and the thickness of the sections to obtain a better resolution. Taking sections at 0.5 lm, we were able to show that in SH-SY5Y and HeLa cells expressing ECFP-labelled spartin within the mitochondria, the tagged protein had a vesicular-like distribution within the

mitochondria, which may suggest an association with the mitochondrial membrane (Figs 2j and l). Occasionally, a spartin-positive structure could be observed that was negative for Mitotracker staining; this was most probably due to the heterogeneity of mitochondria within a single cell, as only mitochondria with a normal membrane potential can effectively take up and store Mitotracker (Chang et al. 2005). Using confocal microscopy in combination with mitochondria-targeted fluorescent proteins and dyes, Collins et al. (2002) have shown that there is morphological and functional heterogeneity of mitochondria even within an individual cell. The C-terminal region contains the primary determinants for mitochondrial localization of spartin In an attempt to define which regions of spartin were associated with mitochondrial localization, we created constructs that would express mutant spartin fused to ECFP. To examine the role of the carboxyl-terminal region, we created an amino terminal-deleted spartin consisting of amino acids 347–666 fused in frame with ECFP (pECFP-SPG20347)666). This protein lacks the MIT domain. We examined the role of amino terminal sequences, including the MIT domain, by examining the localization of two different prematurely truncated spartin constructs. In the first instance, the presence of an inherent EcoRI restriction site within exon 4 of spartin allowed us to create a vector that would express ECFP fused in frame with the first 347 amino acids of spartin (pECFPSPG201)347). We also created a spartin-ECFP construct that mimicked the frameshift mutation seen in patients affected with Troyer syndrome (pECFP-SPG201110delA). To facilitate cloning of this mutant construct it was necessary to introduce a single nucleotide substitution at position 1115 (G to A), which created a MunI restriction site. The effect of the introduced frameshift deletion was identical to that found in Troyer syndrome patients, i.e. substitution of 29 amino acids following the mutation and premature protein truncation so that the expressed protein lacks 268 amino acids (fs369– 398x399). The effect of the single base substitution (G1115A) is the presence of an asparagine residue in place of serine among the substituted amino acids. This substitution should have minimal effect because both are polar noncharged amino acids. These plasmids were transfected into SH-SY5Y and HeLa cells. The N-terminal-deleted ECFP-SPG20347)666 fusion protein had a punctuate expression pattern similar to the pattern observed for full-length spartin (Figs 3a and b), while the prematurely truncated ECFP-SPG201110delA fusion protein was diffusely distributed in the cytoplasm (Figs 3c and d). ECFP-SPG201)347 showed the same diffuse distribution pattern (data not shown). We then used MitoTracker or anti-mitochondrial ATP synthase subunit beta immunocytochemistry on SH-SY5Y and HeLa cells transfected with pECFP-SPG201110delA or pECFP-SPG20347)666 to examine the localization of these mutant spartin fragments in relation

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Fig. 3 Deletion analysis of spartin to map C-terminal sequences important for spartin localisation. (a–d) SH-SY5Y (a, c), mouse primary culture neurone (b) and Neuro2a (d) cells expressing Nterminal-deleted ECFP-SPG20347)666 (a, b) or C-terminal-truncated ECFP-SPG201110delA (c, d) and counterstained with DAPI. Amino terminal-deleted spartin fused to ECFP (ECFP-SPG20347)666) shows a punctuate expression pattern in the cytoplasm, but prematurely truncated spartin (ECFP-SPG201110delA) shows diffuse cytoplasmic expression. (e–l) To investigate mitochondrial localization, MitoTracker (e, g, j–l) or antimitochondria ATP synthase subunit beta

immunocytochemistry (f, h, i) was superimposed on SH-SY5Y (e, f, k), mouse primary culture neurone (g, j) and HeLa (h, i, l) cells expressing amino terminal deleted (ECFP-SPG20347)666) (e–i), or prematurely truncated (ECFP-SPG201110delA) (j–l) spartin. Mitochondrial localization was observed for ECFP-SPG20347)666, while prematurely truncated spartin was distributed diffusely (j–l). In some cells, amino terminal-deleted spartin seemed poorly co-localized with mitochondria (h), but higher magnification (i) showed the ECFP-SPG20347)666 inside the mitochondria (arrow).

to mitochondria. Amino terminal-deleted ECFPSPG20347)666 was found co-localized with mitochondria (Figs 3e–i), while prematurely truncated spartin lacking the C-terminal lost this localization (Figs 3j–l). In some cells, the co-localization of ECFP-SPG20347)666 with mitochondria was not clear-cut, but higher magnification showed that the ECFP-SPG20347)666 did co-localize with mitochondria (Fig. 3i).

Spartin does not localize to endosomes but shows occasional localization to the endoplasmic reticulum To confirm that ECFP-tagged spartin and the derivative C-terminal fragments were located in the mitochondria and not other organelles, we superimposed immunocytochemistry or trackers for endosomes, endoplasmic reticulum and lysosomes (data not shown) in SH-SY5Y and HeLa cells expressing full-length ECFP-tagged spartin, amino

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terminal-deleted spartin tagged with ECFP, or prematurely truncated spartin tagged with ECFP. Though ECFP-SPG20 and ECFP-SPG20347)666 showed a dotty expression and clump-like structures, there was no evidence for localization of spartin to the endosomal marker, anti-EEA1 (Figs 4a, c, e

Fig. 4 Spartin does not show evidence of localization to endosomes or the endoplasmic reticulum. (a-d) SH-SY5Y (a, b) and HeLa (c, d) cells expressing full-length spartin fused to ECFP superimposed with the endosomal marker, anti-EEA1 (a, c) or the endoplasmic reticulum marker, anti-calreticulin (b, d). (e-h) SH-SY5Y (e, f) and HeLa (g, h) cells expressing amino terminal-deleted spartin (ECFP-SPG20347)666) superimposed with the endosomal marker, anti-EEA1 (e, g) or the endoplasmic reticulum marker, anti-calreticulin (f, h). (i, j) SH-SY5Y cells expressing prematurely truncated spartin (ECFP-SPG201)347) superimposed with anti-EEA1 (i) or anti-calreticulin (j).

and g). We did see very slight co-localization of full-length spartin to the endoplasmic reticulum marker, anti-calreticulin, in HeLa cells (Fig. 4d). In cells expressing amino terminal-deleted spartin (ECFP-SPG20347)666) we also saw occasional co-localization with anti-calreticulin (Figs 4f and h). As expected, there was some degree of overlap between endosomal and ER markers with ECFP-SPG201)347 as the latter is diffusely scattered in the cytoplasm (Figs 4i and j). Co-localization of spartin with the microtubule network in neuronal and non-neuronal cells Sequence analysis of spartin identified an MIT domain spanning amino acids 36–114 (Ciccarelli et al. 2003). The MIT domain generally consists of approximately 80 amino acids and it is present in another HSP related protein, spastin, which has been shown to have microtubule-severing activity. Although the MIT domain in spastin is not directly involved in mediating spastin–microtubule interaction, we examined whether spartin displayed evidence of microtubule interaction. We transfected SH-SY5Y and HeLa cells with pECFPSPG20, pECFP-SPG201110delA and pECFP-SPG201)347, which all include the MIT domain, and pECFPSPG20347)666, which does not contain an MIT domain, and counterstained with the anti-alpha tubulin antibody. Our results showed evidence for partial co-localization of fulllength spartin tagged with ECFP with the microtubule network (Figs 5a–c). The prematurely truncated pECFPSPG201)347, which contains the MIT domain, also showed partial co-localization with microtubule structures (Figs 5f and g). We did not observe obvious co-localization of the dotty structure of pECFP-SPG20347)666 with the microtubule network (Figs 5d and e), indicating that the N-terminal region of the protein, which contains the MIT domain, is necessary for the association of spartin with microtubules. While there may appear to be a very small degree of overlap in Fig. 5(e), we believe that the occasional yellow dot is more likely to be due to overlap of the green and red signals than to actual co-localization, because the Z stack of the image cannot be as thin as the microtubule filament. FRET confirmation of spartin–microtubule association We used FRET analysis to confirm the observed association between spartin and microtubules. HeLa cells were co-

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(a)

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Fig. 5 Partial association of full-length and truncated ECFP-spartin with microtubule structure. (a–c) Evidence of partial association of spartin with microtubules seen in SH-SY5Y (a, c) and HeLa (b) cells expressing full-length ECFP-SPG20 superimposed with anti-alphatubulin immunocytochemistry and counterstained with DAPI. (d, e) Association of spartin with microtubules is lost in SH-SY5Y (d) and HeLa (e) cells expressing amino terminal-deleted ECFPSPG20347)666 superimposed with anti-alpha-tubulin immunocytochemistry and counterstained with DAPI. (f, g) SH-SY5Y (f) and HeLa (g) cells expressing prematurely truncated ECFP-SPG201)347 superimposed with anti-alpha-tubulin immunocytochemistry and counterstained with DAPI.

transfected with plasmids expressing full-length spartin fused to ECFP and alpha-tubulin fused to EYFP. We also performed co-transfections of HeLa cells with plasmids

expressing alpha-tubulin fused to EYFP and either amino terminal-deleted spartin (pECFP-SPG20347)666) or prematurely truncated spartin (pECFP-SPG201)347 or pECFPSPG201110delA). Cells expressing both EYFP-tubulin and ECFP-tagged spartin were examined for the appearance of FRET using a single laser acceptor photobleaching method under a confocal microscope. Increased FRET (Ef > 0) indicates that the two fusion proteins are in the vicinity, evidence of interaction between the two proteins (JaresErijman and Jovin 2003). The acceptor photobleaching method has high specificity by photobleaching EYFP before FRET measurement (Kenworthy 2001; Karpova et al. 2003). As a control, cells expressing both ECFP and EYFP showed weak FRET (Ef ¼ 0.9513 ± 0.1825), indicating limited ECFP-EYFP dimer formation which is in accordance with previous reports. As a positive control we used cells expressing ECFP-spastin and EYFP-alpha tubulin. We observed increased FRET efficiency as expected, as spastin interaction with microtubules is well documented (Ef ¼ 11.0254 ± 5.9730). A similar statistically significant increase in FRET efficiency (Ef ¼ 8.007 ± 5.927) was observed when the EYFP of HeLa cells expressing both ECFP-spartin and EYFP-alpha tubulin was photobleached, while there was a decrease in the unbleached area (Cf ¼ ) 3.708 ± 3.455) (Figs 6a and b). In cells co-expressing prematurely truncated spartin, ECFP-SPG201110delA and EYFP-alpha tubulin, photobleaching resulted in only a mild increase in FRET efficiency (Ef ¼ 2.807 ± 4.306) (Figs 6c and d), providing evidence of interaction between the amino terminal region of spartin and alpha-tubulin, albeit at a reduced level when compared with the level observed for full-length spartin and alpha-tubulin. The FRET efficiency of cells co-expressing amino terminal-deleted spartin, ECFP-SPG20347)666 and alpha-tubulin was decreased after photobleaching (Ef ¼ ) 2.449 ± 3.0645) (Figs 6e and f), indicating that amino terminal-deleted spartin had lost the ability to interact with alpha-tubulin. These results indicate that the interaction of spartin with the microtubule network was strongest for fulllength spartin-ECFP, reduced slightly for the prematurely truncated fusion protein and completely lost in amino terminal-deleted spartin. This is perhaps not surprising as the amino terminal-deleted spartin has lost the MIT domain. Discussion

The group of disorders known as HSP represent a clinically and genetically diverse group of disorders characterized pathologically by retrograde axonal degeneration that, in affected patients, manifests as paralysis, spasticity and hyperreflexia, which can be further complicated by additional symptoms. Rapid advances in the functional analysis of defective genes causing HSP suggest that defects in microtubule-mediated vesicle trafficking and/or mitochondrial dysfunction are the causes of axonal degeneration. In this

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Mitochondrial localisation of spartin 1917

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FRET Efficiency

Fig. 6 FRET analysis of spartin microtubule association. HeLa cells expressing ECFP-SPG20 (a, b), ECFP-SPG201110delA (c, d) or ECFP-SPG20347)666 (e, f) and alpha-tubulin fusion proteins. For each figure (a–f), the left panel shows the expression intensity of ECFP-tagged spartin proteins and the right panel shows the expression intensity of EYFP-tagged alpha-tubulin. Boxes indicate the photobleached areas (before bleach: a, c and e; after bleach: b, d and f). In the photobleached areas, there is an obvious increase in ECFP signal (boxed areas on left panels) in cells expressing fulllength spartin (b compared with a). An increase in ECFP signal can also be seen after photobleaching cells expressing ECFP-SPG201110delA (boxed area on left panels of d compared with c). There is a decrease in ECFP signal in photobleached areas in cells expressing ECFPSPG20347)666 (boxed area on left panels of f compared with e). There is a dramatic decrease in EYFP after photobleaching (boxed areas in right panels of b, d and f compared with a, c and e). There is also a noticeable reduction in EYFP signal in general after repeated scanning in the nonphotobleached areas (non- boxed areas of the right panels of b, d and f compared with a, c and e). Statistical analysis (g) showing that FRET efficiency (Ef) of bleached area in ECFP-SPG20 or ECFP-SPG201110delA is increased (p < 0.0001), while those of ECFP-SPG20347)666 and unbleached areas (Cf) are decreased.

5

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study we set out to gain insight into the cellular localization of spartin, a novel protein which, when mutated, causes Troyer syndrome, a form of HSP complicated by the presence of dysarthria, distal amyotrophy, mild developmental delay and short stature. Using confocal microscopy and FRET analysis, we have determined for the first time that spartin is localized in mitochondria and shows an association with microtubules. Furthermore, we have established that a recombinant mutant spartin, engineered to mimic the truncated spartin found in Troyer syndrome patients, has lost its mitochondrial localization. This truncated spartin continues to show an association with microtubules albeit with decreased efficiency. Our observation of mitochondrial localization means that spartin is the third nuclear-encoded mitochondrial protein to be associated with HSP, which further strengthens the importance of mitochondrial function to axonal maintenance.

ECFP-SPG20 EYFP-Tubulin

ECFP-SPG201110delA ECFP-SPG20347-666 EYFP-Tubulin EYFP-Tubulin

The importance of mitochondria ranges from energy supply to the initiation of apoptosis, and there is increasing evidence of a role for mitochondrial dysfunction and oxidative damage in both normal ageing and neurodegenerative diseases (Beal 2005). Paraplegin (SPG7) is a mitochondrial metalloprotease member of the AAA family of proteins. Defects in mitochondrial oxidative phosphorylation are seen in muscle biopsies of HSP patients with mutations in paraplegin (Atorino et al. 2003). Paraplegin is localized to the inner mitochondrial membrane where it forms a large complex with AFG3L2, a homologous protease (Atorino et al. 2003). This complex is found to be aberrant in primary fibroblasts of HSP patients lacking paraplegin, leading to diminished activity of complex I of the respiratory chain and increased sensitivity to oxidative stress that can be rescued by exogenous expression of paraplegin (Atorino et al. 2003). It is thought that the

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paraplegin–AFG3L2 complex has a role in assembling complex I and in the absence of this complex, there is misfolding of proteins and a lack of chaperone activity on complex I assembly. Paraplegin knockout mice demonstrated an axonal accumulation of organelles and neurofilaments, suggesting an impairment of axonal transport (Ferreirinha et al. 2004). A missense mutation was found in the well characterized mitochondrial chaperone, HSP60, in patients with SPG13-linked HSP (Hansen et al. 2002). It is not clear whether the mutant HSP60 acts via a dominant negative mechanism or by haplo-insufficiency leading to reduced overall chaperonin activity. Localization of spartin to the mitochondria is an unexpected finding. The presence of the MIT domain suggested that spartin, like spastin, might localize to endosomes. However, our studies using endosome-specific markers did not show any evidence of endosomal localization. Similarly, spartin showed no evidence of co-localization with lysosomes (data not shown), although we have obtained evidence that spartin occasionally localizes to the endoplasmic reticulum. The majority of mitochondrial proteins are synthesized as precursor proteins carrying cleavable N-terminal targeting signals that direct both the targeting of the proteins to the receptors on the mitochondrial surface and the translocation of proteins across both mitochondrial membranes into the matrix (Neupert 1997). Bioinformatic analysis of spartin showed a weak tendency towards mitochondria targeting (PSORT II 13.0%, MitoProt 0.0175), which could not be expected to account for our results. However, many mitochondrial proteins of the inner and outer membrane lack the Nterminal targeting sequence and instead, contain sorting and targeting information within the mature protein (Rehling et al. 2003). In contrast to easily identifiable N-terminal targeting sequences, the internal signals do not always contain charged amino acid residues and consensus sequences have not been identified (Brix et al. 1999). Most of the outer membrane proteins span the membrane once at either the N- or C-terminal. As our prematurely truncated spartin constructs have lost mitochondrial localization, it is most likely that if spartin resides on the outer membrane, it is a tail-anchored protein with the amino terminal region of spartin exposed to the cytosol. Tail-anchored proteins in the outer mitochondrial membrane, including components of the TOM (translocase of the outer membrane of mitochondria) complex and regulators of apoptosis belonging to the Bcl-2 family, do not share any sequence conservation in their tail region, but all contain a transmembrane domain that is short (16–20 amino acids), moderately hydrophobic and has positively-charged residues at its flanking regions (Rapaport 2003). Bioinformatic analysis of spartin using TMpred (Hofman and Stoffel 1993) to predict transmembrane domains identified three potential transmembrane domains: amino acids 294–313 (TMpred score 879), amino acids 424– 442 (TMpred score 571) and amino acids 537–553 (TMpred

score 1003), where a score greater than 500 indicates a strong transmembrane tendency. We also identified basic amino acid residues and/or mitochondrial targeting signals near the transmembrane sequences. Our results showed that ECFPSPG201)347 and ECFP-SPG201110delA are expressed diffusely in the cytoplasm and have lost their mitochondrial localization, indicating that the transmembrane domain from amino acids 294–328 is not involved in directing spartin to mitochondria. The amino terminal-deleted spartin (ECFPSPG20347)666), which includes two putative internal mitochondrial signals, showed stronger mitochondrial localization. These results are consistent with the transmembrane domains acting as an internal mitochondrial signal. Functional studies of other causative genes in HSP indicate that impaired intracellular trafficking is a major cause of axonal degeneration. Most studies have focused on spastin, which is the most common cause of autosomal dominant (AD) HSP. Interestingly, spastin is the only other HSP-related protein to have an MIT domain. Recent studies show that spastin is a microtubule-severing enzyme, and several disease-associated mutations abolish severing activity (Evans et al. 2005). Spastin interacts with CHMP1B; it also localizes to endosomes (Reid et al. 2005) and endoplasmic reticulum (unpublished data), supporting a role for spastin in intracellular membrane trafficking. Defective transport systems are also implicated in SPG10-linked HSP, where mutations in KIF5A are found (Reid 2003; Fichera et al. 2004). KIF5A encodes a member of the kinesin superfamily that functions as a microtubule motor in intracellular organelle transport. Using confocal microscopy and anti-tubulin antibody, we obtained evidence for spartin association with microtubules. We used FRET analysis to confirm this association, as evidenced by the dramatic increase in FRET efficiency in cells coexpressing ECFP-SPG20 and EYFP-tubulin. As far as we know, this is the first report of spartin interacting with the microtubule network. Our results with ECFP-SPG20, ECFPSPG201110delA and ECFP-SPG201)347 show that sequences in the amino terminal, which include the MIT domain, are essential for microtubule interaction. However, the observed reduction in FRET efficiency in cells co-expressing truncated spartin fused to ECFP (ECFP-SPG201)347) relative to cells expressing full-length spartin indicates that additional sequences are required. A recent study identified Eps15 as a protein interacting with spartin (Bakowska et al. 2005). Eps15 is present in clathrin-coated pits and early and late endosomes, and it is involved in receptor-mediated endocytosis of epidermal growth factor (Torrisi et al. 1999). We did not find evidence of spartin localization to endosomes. Spartin interaction with Eps15 was detected using a yeast two hybrid screen that used a fragment of spartin consisting of amino acids 1–206 as bait. It may be that spartin–Eps15 interaction does not represent a physiological interaction; indeed, the authors were unable to co-immunoprecipitate native Eps15 and spartin.

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Trafficking of vesicles, mitochondria and other organelles along microtubules is crucial for longer axons such as the corticospinal tracts that are affected in HSP, and defects in trafficking systems are likely to have an effect on the more distant ends of the axons. This study reports for the first time the subcellular localization of spartin to mitochondria, and demonstrates partial co-localization and interaction of spartin with microtubules. It is possible that spartin anchors in the mitochondria, and that the exposed N terminal region is free to interact with microtubules. The mutation that has been described in patients with Troyer syndrome is a frameshift mutation leading to premature truncation of spartin. The mutant spartin we constructed to mimic this mutation has lost its mitochondrial localization and shows a reduced affinity for microtubules. It is tempting to speculate that defective intracellular trafficking involving microtubule-related mitochondrial transport is involved in the pathogenesis of Troyer syndrome. It is also possible that spartin has another function in the mitochondria, and the loss of spartin from mitochondria leads to mitochondrial dysfunction and subsequent axonal degeneration. Acknowledgements This work was supported by the Health Research Board of Ireland.

References Atorino L., Silvestri L., Koppen M., Cassina L., Ballabio A., Marconi R., Langer T. and Casari G. (2003) Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia. J. Cell Biol. 163, 777–787. Bakowska J. C., Jenkins R., Pendleton J. and Blackstone C. (2005) The Troyer syndrome (SPG20) protein spartin interacts with Eps15. Biochem. Biophys. Res. Commun. 334, 1042–1048. Beal M. F. (2005) Mitochondria take centre stage in aging and neurodegeneration. Ann. Neurol. 58, 495–505. Brix J., Rudiger S., Bukau B., Schneider-Mergener J. and Pfanner N. (1999) Distribution of binding sequences for the mitochondrial import receptors Tom20, Tom22, and Tom70 in a presequencecarrying preprotein and a non-cleavable preprotein. J. Biol. Chem. 274, 16 522–16 530. Casari G., De Fusco M., Ciarmatori S. et al. (1998) Spastic paraplegia and OXPHOS impairment caused by mutations in paraplegin, a nuclear-encoded mitochondrial metalloprotease. Cell 93, 973–983. Chang S., ran Ma T., Miranda R. D., Balestra M. E., Mahley R. W. and Huang Y. (2005) Lipid- and receptor-binding regions of apolipoprotein E4 fragments act in concert to cause mitochondrial dysfunction and neurotoxicity. Proc. Natl Acad. Sci. USA 102, 18 694–18 699. Ciccarelli F. D., Proukakis C., Patel H., Cross H., Azam S., Patton M. A., Bork P. and Crosby A. H. (2003) The identification of a conserved domain in both spartin and spastin, mutated in hereditary spastic paraplegia. Genomics 81, 437–441. Collins T. J., Berridge M. J., Lipp P. and Bootman M. D. (2002) Mitochondria are morphologically and functionally heterogenous within cells. EMBO J. 21, 1616–1627.

Errico A., Ballabio P. and Rugarli E. (2002) Spastin, the protein mutated in autosomal dominant hereditary spastic paraplegia, is involved in microtubule dynamics. Hum. Mol. Genet. 11, 153–163. Errico A., Claudiani P., D’Addio M. and Rugarli E. I. (2004) Spastin interacts with the centrosomal protein NA14, and is enriched in the spindle pole, the midbody and the distal axon. Hum. Mol. Genet. 13, 2121–2132. Evans K. J., Gomes E. R., Reisenweber S. M., Gundersen G. G. and Lauring B. P. (2005) Linking axonal degeneration to microtubule remodeling by spastin-mediated microtubule severing. J. Cell Biol. 168, 599–606. Ferreirinha F., Quattrini A., Pirozzi M. et al. (2004) Axonal degeneration in paraplegin-deficient mice is associated with abnormal mitochondria and impairment of axonal transport. J. Clin. Invest. 113, 231–242. Fichera M., Lo Giudice M., Falco M., Sturnio M., Amata S., Calabrese O., Bigoni S., Calzolari E. and Neri M. (2004) Evidence of kinesin heavy chain (KIF5A) involvement in pure hereditary spastic paraplegia. Neurology 63, 1108–1110. Fink J. K. (2002) Hereditary spastic paraplegia. Neurol. Clin. 20, 711– 726. Hansen J. J., Durr A., Cournu-Rebeix I. et al. (2002) Hereditary spastic paraplegia SPG13 is associated with a mutation in the gene encoding the mitochondrial chaperonin Hsp60. Am. J. Hum. Genet. 70, 1328–1332. Hazan J., Fonknechten N., Mavel D. et al. (1999) Spastin, a new AAA protein, is altered in the most frequent form of autosomal dominant spastic paraplegia. Nat. Genet. 23, 296–303. Hofman K. and Stoffel W. (1993) TMbase - a database of membrane spanning protein segments. Bio. Chem. Hoppe-Seyler. 374, 166. Jares-Erijman E. A. and Jovin T. M. (2003) FRET imaging. Nat. Biotechnol. 21, 1387–1395. Karpova T. S., Baumann C. T., He L., Wu X., Grammer A., Lipsky P., Hager G. L. and McNally J. G. (2003) Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser. J. Microsc. 209, 56–70. Kenworthy A. K. (2001) Imaging protein–protein interactions using fluorescence resonance energy transfer microscopy. Methods 24, 289–296. Neupert W. (1997) Protein import into mitochondria. Ann. Rev. Biochem. 66, 863–917. Patel H., Cross H., Proukakis C., Hershberger R., Bork P., Ciccarelli F. D., Patton M. A., McKusick V. A. and Crosby A. H. (2002) SPG20 is mutated in Troyer syndrome, a hereditary spastic paraplegia. Nat. Genet. 31, 347–348. Rapaport D. (2003) Finding the right organelle. EMBO Rep. 4, 948–962. Rehling P., Pfanner N. and Meisinger C. (2003) Insertion of hydrophobic membrane proteins into the inner mitochondrial membrane – a guided tour. J. Mol. Biol. 326, 639–657. Reid E. (2003) Science in motion: common molecular pathological themes emerge in the hereditary spastic paraplegias. J. Med. Genet. 40, 81–86. Reid E., Connell J., Edwards T. L., Duley S., Brown S. E. and Sanderson C. M. (2005) The hereditary spastic paraplegia protein interacts with the ESCRT-III complex associated endosomal protein CHMP1B. Hum. Mol. Genet. 14, 19–38. Torrisi M. R., Lotti L. V., Belleudi F., Gradini R., Salcini A. E., Confalonieri S., Pelicci P. G. and Di Fiore P. P. (1999) Eps15 is recruited to the plasma membrane upon epidermal growth factor receptor activation and localizes to components of the endocytic pathway during receptor internalization. Mol. Biol. Cell 10, 417– 434.

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