This work demonstrates that α-synuclein inhibits the biosynthetic secretory pathway of mammalian cells potently and directly under nontoxic conditions and in the absence of insoluble α-synuclein aggregates. A potential mechanism involving α-synuclein binding to ER/Golgi SNAREs and inhibiting fusogenic SNARE complex assembly is elucidated.

Background: Whether ER to Golgi transport requires calcium, the source of calcium, and its mechanism is unknown. Results: A requirement for luminal calcium is demonstrated, and evidence is presented for a molecular effector pathway. Conclusion: Luminal calcium may regulate transport by activating these protein interactions. Significance: The described calcium effector pathway may lead to greater insight into calcium action at multiple transport steps.

Previous studies show that the COPII coat is phosphorylated. The phosphorylated coat, however, cannot rebind to the ER to initiate a new round of vesicle budding. The present study shows that Sit4p/PP6, a Ser/Thr phosphatase, dephosphorylates the COPII coat. Consistent with a role in coat recycling, Sit4p/PP6 regulates ER-to-Golgi traffic.

Trypanosoma congolense is one of the most economically important pathogens of livestock in Africa. Culture-derived parasites of each of the three main insect stages of the T. congolense life cycle, i.e., the procyclic, epimastigote and metacyclic stages, and bloodstream stage parasites isolated from infected mice, were used to construct stage-specific cDNA libraries and expressed sequence tags (ESTs or cDNA clones) in each library were sequenced. Thirteen EST clusters encoding different variant surface glycoproteins (VSGs) were detected in the metacyclic library and twenty-six VSG EST clusters were found in the bloodstream library, six of which are shared by the metacyclic library. Rare VSG ESTs are present in the epimastigote library, and none were detected in the procyclic library. ESTs encoding enzymes that catalyze oxidative phosphorylation and amino acid metabolism are about twice as abundant in the procyclic and epimastigote stages as in the metacyclic and bloodstream stages. In contrast, ESTs encoding enzymes involved in glycolysis, the citric acid cycle and nucleotide metabolism are about the same in all four developmental stages. Cysteine proteases, kinases and phosphatases are the most abundant enzyme groups represented by the ESTs. All four libraries contain T. congolense-specific expressed sequences not present in the T. brucei and T. cruzi genomes. Normalized cDNA libraries were constructed from the metacyclic and bloodstream stages, and found to be further enriched for T. congolense-specific ESTs. Given that cultured T. congolense offers an experimental advantage over other African trypanosome species, these ESTs provide a basis for further investigation of the molecular properties of these four developmental stages, especially the epimastigote and metacyclic stages for which it is difficult to obtain large quantities of organisms. The T. congolense EST databases are available at: http://www.sanger.ac.uk/Projects/T_congolense/EST_index.shtml.

A distinctive feature of Trypanosoma brucei and other trypanosomatids is that most of their genes are transcribed into long polycistronic RNAs [1], which are processed into mature monocistronic mRNAs bearing a 39-nucleotide spliced leader (SL) at their 5′ ends that is derived from a capped precursor RNA of ∼140 nucleotides [2;3]. This trans RNA splicing serves two major functions: in conjunction with 3′ polyadenylation it generates the mature mRNAs from polycistronic primary transcripts, and via the SL it provides a 5′ cap structure for each mRNA [1;4]. In contrast to cis RNA splicing of introns in other eukaryotes, trans RNA splicing unites exons from two independently transcribed RNAs. trans and cis RNA splicing, however, share several similarities. For example, at least some components of the cis RNA spliceosomes are conserved in T. brucei, and both cis and trans RNA splicing utilize the same general mechanism and require the same general sequence motifs [4]. During an examination of the effect of different 3′ UTRs on gene expression in T. brucei, we noticed differences between bloodstream form (BSF) and procyclic form (PCF) trypanosomes in the addition of SLs to RNAs transcribed from a luciferase reporter gene inserted into the rRNA gene locus. Further inspection revealed that in PCF cells the expected SL addition occurs to precursor luciferase RNA, whereas in BSF cells multiple SL additions occur to the same luciferase RNA that are independent of the 5′ and 3′ UTR sequences. Fig. 1A depicts the luciferase reporter plasmid, pMP, integrated into the rRNA gene spacer region of the T. brucei genome that was used for the experiments. This plasmid is derived from plasmid pHD496 (kindly provided by C. Clayton), and is used to measure luciferase activity in BSF and PCF cells when various 3′-UTRs are inserted at a BamHI site directly behind the luciferase coding sequence (LUC). An initial northern blot of RNA from BSF cells bearing integrated pMP revealed the presence of multiple RNA species recognized by a LUC probe (Fig 1B). The three bands indicated by arrowheads coincide with the locations of rRNAs and are likely due to entrapment of some luciferase RNA in these rRNAs. The RNA bands indicated by L (long), LUC and S (short) were examined further. To look for potential differential luciferase expression from the same inserted plasmid in BSF and PCF cells of the same * Corresponding author. Tel.: +1-319-335-7934; fax: +1-319-335-9570, Email address: E-mail: john-donelson@uiowa.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH...