Lewy bodies are intracellular fibrillar inclusions composed of ␣-synuclein. They constitute the pathological hallmark of Parkinson's disease, dementia with Lewy bodies, and other neurodegenerative diseases. Although the majority of Lewy bodies are stained for ubiquitin by immunohistochemistry, the substrate for this modification is poorly understood. Insoluble, urea-soluble ␣-synuclein was separated from soluble fractions and subjected to two-dimensional gel electrophoresis to further characterize pathogenic ␣-synuclein species from disease brains. By using this approach, we found that in sporadic Lewy body diseases a highly modified, diseaseassociated 22-24-kDa ␣-synuclein species is ubiquitinated. Conjugation of one, two, and, to a lesser extent, three ubiquitins was detected. This 22-24-kDa ␣-synuclein species represents partly phosphorylated protein. Furthermore, no generalized impairment of the proteolytic activity of the proteasome was detected in brain regions with Lewy body pathology. Because unmodified ␣-synuclein is degraded by the proteasome in a ubiquitin-independent manner, these data suggest that accumulation of modified 22-24-kDa ␣-synuclein is a disease-specific event which may overwhelm the proteolytic system, leading to aberrant ubiquitination. Accordingly, carboxyl-terminal-truncated ␣-synuclein, presumably the result of aberrant proteolysis, is found only in association with ␣-synuclein aggregates.Lewy bodies (LB) 1 are intracytoplasmic eosinophilic inclusions, which (ultrastructurally) are made of a core of granular and filamentous material surrounded by radially oriented filaments 10 -15 nm in diameter (1). The element of the LB fibril remained unknown until genetic studies in early onset autosomal-dominant Parkinson's disease (PD) led to the identification of two mutations in the ␣-synuclein gene (2, 3). This finding was followed by the identification of ␣-synuclein as the major component of the LB fibrils in sporadic PD and dementia with LB (DLB) (4, 5). LB pathology and ␣-synuclein aggregation in neurons may contribute to their dysfunction and degeneration. Formation of ␣-synuclein fibrils has been studied extensively in in vitro systems using recombinant protein. However, the mechanism by which ␣-synuclein, a natively unfolded protein, accumulates in neurons to form insoluble fibrils with amyloid characteristics is largely unknown. It is feasible that in vivo post-translational modifications interfere with the function and/or degradation of ␣-synuclein or alter its biophysical properties in a way to facilitate aggregation. Alternatively, protein modifications may occur in an attempt to prevent aberrant interactions and/or inhibit further aggregation. Therefore, a detailed understanding of the extent to which disease-associated ␣-synuclein is modified may provide insights into cellular pathways that are activated during fibril formation.In this study, we used differential centrifugation and 2-dimensional gel electrophoresis (2-DE) to characterize LB-associated ␣-synuclein. This approach ...

Amphiphysins 1 and 2 are enriched in the mammalian brain and are proposed to recruit dynamin to sites of endocytosis. Shorter amphiphysin 2 splice variants are also found ubiquitously, with an enrichment in skeletal muscle. At the Drosophila larval neuromuscular junction, amphiphysin is localized postsynaptically and amphiphysin mutants have no major defects in neurotransmission; they are also viable, but flightless. Like mammalian amphiphysin 2 in muscles, Drosophila amphiphysin does not bind clathrin, but can tubulate lipids and is localized on T-tubules. Amphiphysin mutants have a novel phenotype, a severely disorganized T-tubule/sarcoplasmic reticulum system. We therefore propose that muscle amphiphysin is not involved in clathrin-mediated endocytosis, but in the structural organization of the membrane-bound compartments of the excitation-contraction coupling machinery of muscles.

The import of nucleus-encoded proteins into chloroplasts is mediated by translocon complexes in the envelope membranes. A component of the translocon in the outer envelope membrane, Toc34, is encoded in Arabidopsis by two homologous genes, atTOC33 and atTOC34 . Whereas atTOC34 displays relatively uniform expression throughout development, atTOC33 is strongly upregulated in rapidly growing, photosynthetic tissues. To understand the reason for the existence of these two related genes, we characterized the atTOC33 knockout mutant ppi1 . Immunoblotting and proteomics revealed that components of the photosynthetic apparatus are deficient in ppi1 chloroplasts and that nonphotosynthetic chloroplast proteins are unchanged or enriched slightly. Furthermore, DNA array analysis of 3292 transcripts revealed that photosynthetic genes are moderately, but specifically, downregulated in ppi1 . Proteome differences in ppi1 could be correlated with protein import rates: ppi1 chloroplasts imported the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit and 33-kD oxygen-evolving complex precursors at significantly reduced rates, but the import of a 50S ribosomal subunit precursor was largely unaffected. The ppi1 import defect occurred at the level of preprotein binding, which is consistent with a role for atToc33 during preprotein recognition. The data suggest that atToc33 is involved preferentially in the import of photosynthetic proteins and, by extension, that atToc34 is involved in the import of nonphotosynthetic chloroplast proteins.

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Phosphatidic acid (PA) is postulated to have both structural and signaling functions during membrane dynamics in animal cells. In this study, we show that before a critical time period during rhabdomere biogenesis in Drosophila melanogaster photoreceptors, elevated levels of PA disrupt membrane transport to the apical domain. Lipidomic analysis shows that this effect is associated with an increase in the abundance of a single, relatively minor molecular species of PA. These transport defects are dependent on the activation state of Arf1. Transport defects via PA generated by phospholipase D require the activity of type I phosphatidylinositol (PI) 4 phosphate 5 kinase, are phenocopied by knockdown of PI 4 kinase, and are associated with normal endoplasmic reticulum to Golgi transport. We propose that PA levels are critical for apical membrane transport events required for rhabdomere biogenesis.