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.

In the Drosophila flight muscle actin mutant E93K there is a charge reversal on the surface of actin close to the proposed position of tropomyosin when it is in the off state. Using a quantitative in vitro motility assay we have found that the wild type Drosophila ACT88F actin behaved like rabbit skeletal muscle actin when tropomyosin and troponin were added at pCa5 and pCa9. In contrast the effect of tropomyosin upon the E93K mutant actin filament movement was completely different from wild type and resembled the response of wild type with tropomyosin؉troponin at pCa9 (i.e. the filaments were switched off). Velocity of E93K actin did not increase, and the fraction of filaments motile was reduced to less than 15% by adding up to 30 nM tropomyosin. When myosin subfragment-1 modified by N-ethylmaleimide was mixed with mutant E93K actin-tropomyosin filaments we observed that it restored motility of the filaments to the level observed with E93K actin alone. We conclude that electrostatic charge on the surface of domain 2 of actin plays a critical role in determining the state of actin-tropomyosin that is a central feature of the steric blocking mechanism of actin filament regulation.

Many mutants have been described that affect the function of the actin encoded by the Drosophila melanogaster indirect flight muscle-specific actin gene, Act88F. We describe the development of procedures for purification of this actin from the other isoforms expressed in the fly as well as in vitro motility, single molecule force/ displacement measurements, and stop-flow solution kinetic studies of the wild-type actin and that of the E93K mutation of the Act88F gene. We show that this mutation affects in vitro motility of F-actin, in both the presence and absence of methylcellulose, and the ability of the ACT88F actin to bind the S1 fragment of rabbit skeletal myosin. However, optical tweezer measurements of the actomyosin working stroke and the force transmitted from the rabbit heavy meromyosin to and through Factin are unchanged by the mutation. These results support the proposal (Holmes, K. C. (1995) Biophys J. 68, (suppl.) 2-7) that actin residue Glu 93 is part of the secondary myosin binding site and suggest that myosin binding occurs first at the primary myosin binding site and then at the secondary site.Actin and myosin are ubiquitous in eukaryotic cells, where they form one of the major motor protein systems. The atomic structure determinations of the actin monomer (2) and the S1 fragment (motor domain) of the chicken skeletal muscle myosin (3) were major landmarks in our understanding of how this motor system works.

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