Interaction of the SPG21 protein ACP33/maspardin with the aldehyde dehydrogenase ALDH16A1 - PubMed (original) (raw)

Interaction of the SPG21 protein ACP33/maspardin with the aldehyde dehydrogenase ALDH16A1

Michael C Hanna et al. Neurogenetics. 2009 Jul.

Abstract

Mast syndrome (SPG21) is an autosomal-recessive complicated form of hereditary spastic paraplegia characterized by dementia, thin corpus callosum, white matter abnormalities, and cerebellar and extrapyramidal signs in addition to spastic paraparesis. A nucleotide insertion resulting in premature truncation of the SPG21 gene product acidic cluster protein 33 (ACP33)/maspardin underlies this disorder, likely causing loss of protein function. However, little is known about the function of maspardin. Here, we report that maspardin localizes prominently to cytoplasm as well as to membranes, possibly at trans-Golgi network/late endosomal compartments. Immunoprecipitation of maspardin with identification of coprecipitating proteins by mass spectrometry revealed the aldehyde dehydrogenase ALDH16A1 as an interacting protein. This interaction was confirmed using overexpressed proteins as well as by fusion protein pull down experiments, and these proteins colocalized in cells. Further studies of the function of ALDH16A1 and the role of the maspardin-ALDH16A1 interaction in neuronal cells may clarify the cellular pathogenesis of Mast syndrome.

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Figures

Fig. 1

Fig. 1

Characterization of polyclonal anti-maspardin antibodies. a, b Lysates from mock-transfected (Untrans; 30 μg protein per lane) or maspardin-transfected (3 μg protein) HeLa cells were immunoblotted with antibodies against N- (a) or C-terminal (b) regions of maspardin. Migration of molecular mass standards (in kilodalton) are shown to the left. c, d HeLa cells were immunostained using N-terminal (c) or C-terminal (d) antibodies, with detection by confocal microscopy. eh Immunodetection for both N- and C-terminal antibodies, respectively, could be eliminated on immunoblots (e, f) and immunostaining (g, h) by preadsorption with the antigenic peptides

Fig. 2

Fig. 2

Subcellular distributions of maspardin in HeLa cells. a Fractionation of HeLa cell lysates was performed using the Qiagen Qproteome Cell Compartment Kit. Fractions (20 μg protein per lane) were immunoblotted with antibodies against maspardin (no. c3945), the cytoplasmic protein PLC-γ, the endoplasmic reticulum protein calnexin, and the nuclear envelope protein lamin A + C. Abbreviations: C, cytosolic fraction; M, membrane fraction; N, nuclear fraction. b Subcellular fractions were prepared from HeLa cells by differential centrifugation. Fractions (30 μg of protein per lane) were immunoblotted with antibodies against maspardin, the mitochondrial protein OPA1, calnexin, and PLC-γ. Specific fractions are described in the “Materials and methods.” Abbreviation: H, homogenate. c Detergent-solubilized extracts prepared from HeLa cells were subjected to FPLC gel-exclusion chromatography, and aliquots of collected fractions were immunoblotted with anti-maspardin antibodies. Elution peaks for marker proteins (in kilodalton) are indicated across the top

Fig. 3

Fig. 3

Subcellular localization of maspardin. HeLa cells were coimmunostained for endogenous maspardin (red) and the indicated marker proteins (a_–_f, green). Merged images are at the right. Abbreviation: M6P, man-nose-6-phosphate receptor. Bars, 10 μm

Fig. 4

Fig. 4

Identification of maspardin-associated proteins in HeLa cells. HeLa cell lysates or else extraction buffer alone was immunoprecipitated using an anti-maspardin antibody (no. c3945), and in control experiments HeLa cells extracts were immunoprecipitated with nonimmune IgG. Proteins coimmunoprecipitating with maspardin were resolved on 10% SDS-PAGE gels and stained with MALDI-TOF-mass-spectroscopy-compatible Coomassie blue. Only proteins present specifically in HeLa + c3945, and not in the nonspecific binding control conditions, were excised for enzymatic digestion by trypsin and identification by mass spectrometry. Maspardin (~33 kDa, arrow) and ALDH16A1 (~80 kDa, arrow) were clearly identified. Other coimmunoprecipitating proteins are evident but have not been independently verified (arrowheads). Heavy and light chains of IgG are indicated

Fig. 5

Fig. 5

Maspardin associates with ALDH16A1 in HeLa cells. a, b HeLa cells were mock-transfected or else transfected with full-length ALDH16A1, epitope-tagged as indicated. Extracts were immunoprecipitated with control IgG (−IP) or polyclonal anti-maspardin antibodies to either the N-terminal (b5704) or the C-terminal (c3945) region and then immunoblotted with anti-Myc (a) or anti-HA (b) monoclonal antibodies to detect ALDH16A1. c Reciprocal immunoprecipitation experiments in which endogenous ALDH16A1 was immunoprecipitated using polyclonal anti-ALDH16A1 antibodies and then immunoblotted with anti-Myc antibodies to detect overexpressed Myc-maspardin. d Untransfected HeLa cell lysates or else control lysis buffer were immunoprecipitated with control IgG (−IP) or polyclonal anti-maspardin antibodies and immunoblotted with polyclonal anti-ALDH16A1 antibodies. e Confocal fluorescence microscopy of endogenous ALDH16A1 and maspardin demonstrate similar subcellular localizations within HeLa cells and distinct regions of colocalization (merge). Immuno-reactive signals were greatly reduced by preadsorption of the antibodies with their respective antigenic peptides. Bars, 10 μm

Fig. 6

Fig. 6

Specificity of maspardin–ALDH16A1 interactions. a Maspardin interacts with ALDH16A1 in vitro. Immobilized GST–maspardin or control GST were used in pull down assays with purified CBP–ALDH16A1. Bound proteins were resolved by SDS-PAGE and immunoblotted with antibodies against CBP. b Lysates from wild-type Myc-maspardin (WT) or Myc-maspardin-S109A-transfected HeLa cells were immunoprecipitated using anti-Myc antibodies or control mouse IgG and then immunoblotted for ALDH16A1. Endogenous ALDH16A1 is not visible in the input lanes at this exposure but is enriched in anti-Myc immunoprecipitates. c Extracts from untransfected or Myc-ALDH16A1-transfected cells were immunoprecipitated with control IgG or anti-maspardin antibodies (Maspardin IP) and immunoblotted with anti-ALDH1A1 antibodies. In control experiments; protein A–Sepharose beads (Beads) alone were added without immunoglobulins. Migrations of the ALDH1A1 protein and IgG heavy chain are indicated. Migrations of molecular mass standards (in kilodalton) are shown to the right. In all panels, inputs represent 10% of the total lysate

Fig. 7

Fig. 7

Maspardin and ALDH16A1 interactions in the central nervous system. a RT-PCR reaction products from total RNA extracted from mouse cortex. Sizes are 511 bp for maspardin and 512 bp for ALDH16A1. Full-length cDNA of maspardin and ALDH16A1 serve as both positive and negative controls, as shown. b Untransfected HeLa cell and mouse brain tissue lysates were immunoprecipitated with control IgG (for brain lysates only) or anti-maspardin antibodies and then immunoblotted with anti-ALDH16A1 antibodies. Where indicated (Buffer), no cell extracts were added. c Cultured primary cortical neurons were costained with antibodies against maspardin (green) and ALDH16A1 (red) and imaged using confocal microscopy. Boxed areas in the phase-contrast superimposed image on the left are enlarged on the right

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