Troyer syndrome protein spartin is mono-ubiquitinated and functions in EGF receptor trafficking

Troyer syndrome is an autosomal recessive hereditary spastic paraplegia caused by mutation in the spartin (SPG20) gene, which encodes a widely expressed protein of unknown function. This mutation results in premature protein truncation and thus might signify a loss-of-function disease mechanism. In this study, we have found that spartin is mono-ubiquitinated and functions in degradation of the epidermal growth factor receptor (EGFR). Upon EGF stimulation, spartin translocates from the cytoplasm to the plasma membrane and colocalizes with internalized EGF-Alexa. Knockdown of spartin by small interfering RNA decreases the rate of EGFR degradation and also affects EGFR internalization, recycling, or both. Furthermore, overexpression of spartin results in a prominent decrease in EGFR degradation. Taken together, our data suggest that spartin is involved in the intracellular trafficking of EGFR and that impaired endocytosis may underlie the pathogenesis of Troyer syndrome.     

The hereditary spastic paraplegia protein spartin localises to mitochondria

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 microtubule-mediated trafficking of mitochondria and/or mitochondrial dysfunction.     

SPG20 protein spartin associates with cardiolipin via its plant-related senescence domain and regulates mitochondrial Ca2+ homeostasis

Hereditary spastic paraplegias (HSPs) are a group of neurological disorders characterized clinically by spasticity of lower limbs and pathologically by degeneration of the corticospinal tract. Troyer syndrome is an autosomal recessive HSP caused by a frameshift mutation in the spartin (SPG20) gene. Previously, we established that this mutation results in a lack of expression of the truncated mutant spartin protein. Spartin is involved in many cellular processes and associates with several intracellular organelles, including mitochondria. Spartin contains a conserved plant-related senescence domain at its C-terminus. However, neither the function of this domain nor the roles of spartin in mitochondrial physiology are currently known. In this study, we determined that the plant-related senescence domain of spartin interacts with cardiolipin but not with two other major mitochondrial phospholipids, phosphatidylcholine and phosphatidylethanolamine. We also found that knockdown of spartin by small interfering RNA in a human neuroblastoma cell line resulted in depolarization of the mitochondrial membrane. In addition, depletion of spartin resulted in a significant decrease in both mitochondrial calcium uptake and mitochondrial membrane potential in cells treated with thapsigargin. Our results suggest that impairment of mitochondrial calcium uptake might contribute to the neurodegeneration of long corticospinal axons and the pathophysiology of Troyer syndrome.     

Identification of novel spartin-interactors shows spartin is a multifunctional protein

Hereditary spastic paraplegia describes a group of neurodegenerative diseases characterized by lower limb progressive weakness and spasticity. Troyer syndrome is an autosomal recessive form of hereditary spastic paraplegia caused by a frameshift mutation (1110delA) in the SPG20 gene encoding spartin protein, the cellular function of which remains unknown. Knowledge about spartin-interactors is also very limited. In this study, we apply a broad spectrum of proteomics techniques to identify novel spartin-binding proteins. We used a Tandem Affinity Purification technique followed by HPLC-mass spectrometry to characterize potential spartin-binding partners. Selected putative interactions were confirmed by co-immunoprecipitation experiments. We identified 94 potential spartin-binding proteins which were grouped into functional categories. We performed co-immunoprecipitation experiments to confirm that spartin interacts with GRP78, GRP75 and nucleolin proteins. Additionally, our mass spectrometry results confirmed previously published information about spartin interaction with ubiquitin and the E3 ubiquitin-protein ligases, AIP4/Itch and AIP5/WWP1. Our studies suggest that spartin is a multifunctional protein and for the first time we suggest a role for spartin in protein folding and turnover both in mitochondria and endoplasmic reticulum. We also show for the first time interaction between spartin and a nucleolar protein, nucleolin.     

SPG20 Protein Spartin Is Recruited to Midbodies by ESCRT-III Protein Ist1 and Participates in Cytokinesis

Hereditary spastic paraplegias (HSPs, SPG1-46) are inherited neurological disorders characterized by lower extremity spastic weakness. Loss-of-function SPG20 gene mutations cause an autosomal recessive HSP known as Troyer syndrome. The SPG20 protein spartin localizes to lipid droplets and endosomes, and it interacts with tail interacting protein 47 (TIP47) as well as the ubiquitin E3 ligases atrophin-1-interacting protein (AIP)4 and AIP5. Spartin harbors a domain contained within microtubule-interacting and trafficking molecules (MIT) at its N-terminus, and most proteins with MIT domains interact with specific ESCRT-III proteins. Using yeast two-hybrid and in vitro surface plasmon resonance assays, we demonstrate that the spartin MIT domain binds with micromolar affinity to the endosomal sorting complex required for transport (ESCRT)-III protein increased sodium tolerance (Ist)1 but not to ESCRT-III proteins charged multivesicular body proteins 1–7. Spartin colocalizes with Ist1 at the midbody, and depletion of Ist1 in cells by small interfering RNA significantly decreases the number of cells where spartin is present at midbodies. Depletion of spartin does not affect Ist1 localization to midbodies but markedly impairs cytokinesis. A structure-based amino acid substitution in the spartin MIT domain (F24D) blocks the spartin–Ist1 interaction. Spartin F24D does not localize to the midbody and acts in a dominant-negative manner to impair cytokinesis. These data suggest that Ist1 interaction is important for spartin recruitment to the midbody and that spartin participates in cytokinesis.     

The role of spartin and its novel ubiquitin binding region in DALIS occurrence

Troyer syndrome is an autosomal recessive hereditary spastic paraplegia (HSP) caused by frameshift mutations in the SPG20 gene that results in a lack of expression of the truncated protein. Spartin is a multifunctional protein, yet only two conserved domains—a microtubule-interacting and trafficking domain and a plant-related senescence domain involved in cytokinesis and mitochondrial physiology, respectively—have been defined. We have shown that overexpressed spartin binds to the Ile44 hydrophobic pocket of ubiquitin, suggesting spartin might contain a ubiquitin-binding domain. In the present study, we demonstrate that spartin contributes to the formation of dendritic aggresome-like induced structures (DALIS) through a unique ubiquitin-binding region (UBR). Using short hairpin RNA, we knocked down spartin in RAW264.7 cells and found that DALIS frequency decreased; conversely, overexpression of spartin increased the percentage of cells containing DALIS. Using nuclear magnetic resonance spectroscopy, we characterized spartin''s UBR and defined the UBR''s amino acids that are key for ubiquitin binding. We also found that spartin, via the UBR, binds Lys-63–linked ubiquitin chains but does not bind Lys-48–linked ubiquitin chains. Finally, we demonstrate that spartin''s role in DALIS formation depends on key residues within its UBR.     

Spartin: At the crossroad between ubiquitination and metabolism in cancer

SPART is a gene coding for a multifunctional protein called spartin, localized in various organelles of human cells. Mutations in the coding region are responsible for a hereditary form of spastic paraplegia called Troyer syndrome while the epigenetic silencing has been demonstrated for some types of tumors. The main functions of this gene are associated to endosomic trafficking and receptor degradation, microtubule interaction, cytokinesis, fatty acids and oxidative metabolism. Spartin has been shown to be a target regulated by STAT3 and localizes also at the level of the mitochondrial outer membrane, where it forms part of a complex maintaining the integrity of the membrane potential. The most recent evidences report a downregulation of spartin in tumor tissues when compared to adjacent normal samples. This intriguing evidence supports further research aimed at clarifying the role of this protein in cancer development and metabolism.     

Spartin recruits PKC-ζ via the PKC-ζ-interacting proteins ZIP1 and ZIP3 to lipid droplets

Protein kinase C-ζ interacting proteins (ZIP1-3) recruit the enzymatic activity of the atypical protein kinase C isoforms PKC-λ/ι or PKC-ζ to target proteins. In this study, we searched for binding partners of ZIP3 in the CNS and identified spartin, a multifunctional protein that is mutated in spastic paraplegia type 20. In transfected cells, spartin was present on the surface of lipid droplets (LD), whereas ZIP proteins appeared in intracellular speckles. In the presence of spartin, ZIP1 and ZIP3 were translocated to spartin-positive LD. This translocation was mediated by amino acids 196-393 of spartin that interacted with an N-terminal region of ZIP proteins. Furthermore, ZIP proteins interacted simultaneously with spartin and PKC-ζ, resulting in an enrichment of PKC-ζ on spartin/ZIP-labelled LD. Without spartin, neither ZIP proteins nor PKC-ζ were detected on LD. Interestingly, the presence of the spartin/ZIP/PKC-ζ complex increased LD size. This effect was most pronounced upon incorporation of the ZIP3 isoform into the trimer. Finally, we co-localized spartin, ZIP proteins and PKC-ζ in axon terminals of neurons in the mammalian retina. In summary, we describe spartin as new binding partner of the ZIP/PKC-ζ dimer that recruits PKC-ζ to LD and show that the expressed ZIP isoform regulates LD size.     

Regulation of lipid droplet turnover by ubiquitin ligases

Mutation of the protein spartin is a cause of one form of spastic paraplegia. Spartin interacts with ubiquitin ligases of the Nedd4 family, and a recent report in BMC Biology now shows that it acts as an adaptor to recruit and activate the ubiquitin ligase AIP4 onto lipid droplets, leading to the ubiquitination and degradation of droplet-associated proteins. A deficiency of spartin apparently causes lipid droplets to accumulate.     

Endogenous spartin, mutated in hereditary spastic paraplegia, has a complex subcellular localization suggesting diverse roles in neurons

Mutation of spartin (SPG20) underlies a complicated form of hereditary spastic paraplegia, a disorder principally defined by the degeneration of upper motor neurons. Using a polyclonal antibody against spartin to gain insight into the function of the endogenous molecule, we show that the endogenous molecule is present in two main isoforms of 85 kDa and 100 kDa, and 75 kDa and 85 kDa in human and murine, respectively, with restricted subcellular localization. Immunohistochemical studies on human and mouse embryo sections and in vitro cell studies indicate that spartin is likely to possess both nuclear and cytoplasmic functions. The nuclear expression of spartin closely mirrors that of the snRNP (small nuclear ribonucleoprotein) marker alpha-Sm, a component of the spliceosome. Spartin is also enriched at the centrosome within mitotic structures. Notably we show that spartin protein undergoes dynamic positional changes in differentiating human SH-SY5Y cells. In undifferentiated non-neuronal cells, spartin displays a nuclear and diffuse cytosolic profile, whereas spartin transiently accumulates in the trans-Golgi network and subsequently decorates discrete puncta along neurites in terminally differentiated neuroblastic cells. Investigation of these spartin-positive vesicles reveals that a large proportion colocalizes with the synaptic vesicle marker synaptotagmin. Spartin is also enriched in synaptic-like structures and in synaptic vesicle-enriched fraction.     

A role for ubiquitin ligases and Spartin/SPG20 in lipid droplet turnover

HECT (homologous to the E6AP C terminus) ubiquitin ligases have diverse functions in eukaryotic cells. In screens for proteins that bind to the HECT ubiquitin ligase WWP1, we identified Spartin, which is also known as SPG20. This protein is truncated in a neurological disease, Troyer syndrome. In this study, we show that SPG20 associates with the surface of lipid droplets (LDs) and can regulate their size and number. SPG20 binds to another LD protein, TIP47, and both proteins compete with an additional LD protein, adipophilin/adipocyte differentiation-related protein, for occupancy of LDs. The mutant SPG20 present in Troyer syndrome does not possess these activities. Depletion of SPG20 using RNA interference increases the number and size of LDs when cells are fed with oleic acid. Binding of WWP1 to SPG20 and the consequent ubiquitin transfer remove SPG20 from LDs and reduce the levels of coexpressed SPG20. These experiments suggest functions for ubiquitin ligases and SPG20 in the regulation of LD turnover and potential pathological mechanisms in Troyer syndrome.     

Neuropathy target esterase gene mutations cause motor neuron disease

The possibility that organophosphorus (OP) compounds contribute to motor neuron disease (MND) is supported by association of paraoxonase 1 polymorphisms with amyotrophic lateral sclerosis (ALS) and the occurrence of MND in OP compound-induced delayed neuropathy (OPIDN), in which neuropathy target esterase (NTE) is inhibited by organophosphorylation. We evaluated a consanguineous kindred and a genetically unrelated nonconsanguineous kindred in which affected subjects exhibited progressive spastic paraplegia and distal muscle wasting. Affected subjects resembled those with OPIDN and those with Troyer Syndrome due to SPG20/spartin gene mutation (excluded by genetic linkage and SPG20/spartin sequence analysis). Genome-wide analysis suggested linkage to a 22 cM homozygous locus (D19S565 to D19S884, maximum multipoint LOD score 3.28) on chromosome 19p13 to which NTE had been mapped (GenBank AJ004832). NTE was a candidate because of its role in OPIDN and the similarity of our patients to those with OPIDN. Affected subjects in the consanguineous kindred were homozygous for disease-specific NTE mutation c.3034A-->G that disrupted an interspecies conserved residue (M1012V) in NTE's catalytic domain. Affected subjects in the nonconsanguineous family were compound heterozygotes: one allele had c.2669G-->A mutation, which disrupts an interspecies conserved residue in NTE's catalytic domain (R890H), and the other allele had an insertion (c.2946_2947insCAGC) causing frameshift and protein truncation (p.S982fs1019). Disease-specific, nonconserved NTE mutations in unrelated MND patients indicates NTE's importance in maintaining axonal integrity, raises the possibility that NTE pathway disturbances contribute to other MNDs including ALS, and supports the role of NTE abnormalities in axonopathy produced by neuropathic OP compounds.     

Spartin activates atrophin-1-interacting protein 4 (AIP4) E3 ubiquitin ligase and promotes ubiquitination of adipophilin on lipid droplets

Background  

Spartin protein is involved in degradation of epidermal growth factor receptor and turnover of lipid droplets and a lack of expression of this protein is responsible for hereditary spastic paraplegia type 20 (SPG20). Spartin is a multifunctional protein that associates with many cellular organelles, including lipid droplets. Recent studies showed that spartin interacts with E3 ubiquitin ligases that belong to the neural precursor cell-expressed developmentally downregulated gene (Nedd4) family, including atrophin-1-interacting protein 4 (AIP4/ITCH). However, the biological importance of the spartin-AIP4 interaction remains unknown.     

Oxidative Stress in Caenorhabditis elegans: Protective Effects of Spartin

Troyer syndrome is caused by a mutation in the SPG20 gene, which results in complete loss of expression of the protein spartin. We generated a genetic model of Troyer syndrome in worms to explore the locomotor consequences of a null mutation of the Caenorhabditis elegans SPG20 orthologue, F57B10.9, also known as spg-20. Spg-20 mutants showed decreased length, crawling speed, and thrashing frequency, and had a shorter lifespan than wild-type animals. These results suggest an age-dependent decline in motor function in mutant animals. The drug paraquat was used to induce oxidative stress for 4 days in the animals. We measured survival rate and examined locomotion by measuring crawling speed and thrashing frequency. After 4 days of paraquat exposure, 77% of wild-type animals survived, but only 38% of spg-20 mutant animals survived. Conversely, animals overexpressing spg-20 had a survival rate of 95%. We also tested lifespan after a 1 hour exposure to sodium azide. After a 24 hour recovery period, 87% of wild type animals survived, 57% of spg-20 mutant animals survived, and 82% of animals overexpressing spg-20 survived. In the behavioral assays, spg-20 mutant animals showed a significant decrease in both crawling speed and thrashing frequency compared with wild-type animals. Importantly, the locomotor phenotype for both crawling and thrashing was rescued in animals overexpressing spg-20. The animals overexpressing spg-20 had crawling speeds and thrashing frequencies similar to those of wild-type animals. These data suggest that the protein F57B10.9/SPG-20 might have a protective role against oxidative stress.     

PrPc on the road: trafficking of the cellular prion protein

The glycosylphosphatidylinositol (GPI)-anchored cellular prion protein (PrPc) has a fundamental role in prion diseases. Intracellular trafficking of PrPc is important in the generation of protease resistant PrP species but little is known of how endocytosis affects PrPc function. Here, we discuss recent experiments that have illuminated how PrPc is internalized and what are the possible destinations taken by the protein. Contrary to what would be expected for a GPI-anchored protein there is increasing evidence that clathrin-mediated endocytosis and classical endocytic organelles participate in PrPc trafficking. Moreover, the N-terminal domain of PrPc may be involved in sorting events that can direct the protein during its intracellular journey. Indeed, the concept that the GPI-anchor determines PrPc trafficking has been challenged. Cellular signaling can be triggered or be regulated by PrPc and we suggest that endocytosis of PrPc may influence signaling in several ways. Definition of the processes that participate in PrPc endocytosis and intracellular trafficking can have a major impact on our understanding of the mechanisms involved in PrPc function and conversion to protease resistant conformations.     

Pak1 Phosphorylation Enhances Cortactin–N‐WASP Interaction in Clathrin‐Caveolin‐Independent Endocytosis

Growing evidence indicates that kinases are central to the regulation of endocytic pathways. Previously, we identified p21‐activated kinase 1 (Pak1) as the first specific regulator of clathrin‐ and caveolae‐independent endocytosis used by the interleukin 2 receptor subunit (IL‐2R). Here, we address the mechanism by which Pak1 regulates IL‐2Rβ endocytosis. First, we show that Pak1 phosphorylates an activator of actin polymerization, cortactin, on its serine residues 405 and 418. Consistently, we observe a specific inhibition of IL‐2Rβ endocytosis when cells overexpress a cortactin, wherein these serine residues have been mutated. In addition, we show that the actin polymerization enhancer, neuronal Wiskott–Aldrich syndrome protein (N‐WASP), is involved in IL‐2Rβ endocytosis. Strikingly, we find that Pak1 phosphorylation of cortactin on serine residues 405 and 418 increases its association with N‐WASP. Thus, Pak1, by controlling the interaction between cortactin and N‐WASP, could regulate the polymerization of actin during clathrin‐independent endocytosis.     

A Wiskott–Aldrich syndrome protein is involved in endocytosis in Aspergillus nidulans

Endocytosis is vital for hyphal tip growth in filamentous fungi and is involved in the tip localization of various membrane proteins. To investigate the function of a Wiskott–Aldrich syndrome protein (WASP) in endocytosis of filamentous fungi, we identified a WASP ortholog-encoding gene, wspA, in Aspergillus nidulans and characterized it. The wspA product, WspA, localized to the tips of germ tubes during germination and actin rings in the subapical regions of mature hyphae. wspA is essential for the growth and functioned in the polarity establishment and maintenance during germination of conidia. We also investigated its function in endocytosis and revealed that endocytosis of SynA, a synaptobrevin ortholog that is known to be endocytosed at the subapical regions of hyphal tips in A. nidulans, did not occur when wspA expression was repressed. These results suggest that WspA plays roles in endocytosis at hyphal tips and polarity establishment during germination.     

Rab39, a novel Golgi-associated Rab GTPase from human dendritic cells involved in cellular endocytosis

Rab GTPases are Ras-like small molecular weight GTP binding proteins that are involved in various steps along the exocytic and endocytic pathways. Here we report that Rab39, a novel Rab protein, is a Golgi-associated protein involved in endocytosis of HeLa cells. Full-length cDNA of Rab39 contains 1251bp with an open reading frame (ORF) of 636bp, which is predicted to encode a 211 aa protein. By blast analysis of Rab39 cDNA and protein sequence with homologues, we find that Rab39 may be a short variant of Rab34. Rab39 contains conserved motifs involved in phosphate/guanosine binding and a microbody C-terminal targeting signal. RT-PCR analysis indicates that Rab39 is mainly detected in epithelial cell lines, and Northern blot analysis shows that Rab39 is expressed ubiquitously in human tissues. By using FITC-BSA as an endocytic tracer, we show that Rab39 can facilitate endocytosis in HeLa cells when expressed either transiently or stably. Confocal microscopy examination of Rab39 subcellular localization suggests that Rab39 is associated with Golgi-associated organelles. Our findings demonstrate that Rab39 is a novel Rab GTPase involved in cellular endocytosis.