The endomembrane system is a complex and dynamic intracellular trafficking network. It is very challenging to track individual vesicles and their cargos in real time; however, affinity purification allows vesicles to be isolated in their natural state so that their constituent proteins can be identified. Pioneering this approach in plants, we isolated the SYP61 trans-Golgi network compartment and carried out a comprehensive proteomic analysis of its contents with only minimal interference from other organelles. The proteome of SYP61 revealed the association of proteins of unknown function that have previously not been ascribed to this compartment. We identified a complete SYP61 SNARE complex, including regulatory proteins and validated the proteome data by showing that several of these proteins associated with SYP61 in planta. We further identified the SYP121-complex and cellulose synthases, suggesting that SYP61 plays a role in the exocytic trafficking and the transport of cell wall components to the plasma membrane. The presence of proteins of unknown function in the SYP61 proteome including ECHIDNA offers the opportunity to identify novel trafficking components and cargos. The affinity purification of plant vesicles in their natural state provides a basis for further analysis and dissection of complex endomembrane networks. The approach is widely applicable and can afford the study of several vesicle populations in plants, which can be compared with the SYP61 vesicle proteome.

-GlcNAc is a regulatory post-translational modification of nucleocytoplasmic proteins that has been implicated in multiple biological processes, including transcription. In humans, single genes encode enzymes for its attachment (-GlcNAc transferase (OGT)) and removal (-GlcNAcase (OGA)). An X-chromosome exome screen identified a missense mutation, which encodes an amino acid in the tetratricopeptide repeat, in (759G>T (p.L254F)) that segregates with X-linked intellectual disability (XLID) in an affected family. A decrease in steady-state OGT protein levels was observed in isolated lymphoblastoid cell lines from affected individuals, consistent with molecular modeling experiments. Recombinant expression of L254F-OGT demonstrated that the enzyme is active as both a glycosyltransferase and an HCF-1 protease. Despite the reduction in OGT levels seen in the L254F-OGT individual cells, we observed that steady-state global-GlcNAc levels remained grossly unaltered. Surprisingly, lymphoblastoids from affected individuals displayed a marked decrease in steady-state OGA protein and mRNA levels. We observed an enrichment of the OGT-containing transcriptional repressor complex mSin3A-HDAC1 at the proximal promoter region of and correspondingly decreased promoter activity in affected cells. Global transcriptome analysis of L254F-OGT lymphoblastoids compared with controls revealed a small subset of genes that are differentially expressed. Thus, we have begun to unravel the molecular consequences of the 759G>T (p.L254F) mutation in that uncovered a compensation mechanism, albeit imperfect, given the phenotype of affected individuals, to maintain steady-state-GlcNAc levels. Thus, a single amino acid substitution in the regulatory domain (the tetratricopeptide repeat domain) of OGT, which catalyzes the -GlcNAc post-translational modification of nuclear and cytosolic proteins, appears causal for XLID.

Unlike other eukaryotes, the protein-coding genes of Trypanosoma cruzi are arranged in large polycistronic gene clusters transcribed by polymerase II (Pol II). Thus, it is thought that trypanosomes rely solely on posttranscriptional processes to regulate gene expression. Here, we show that the glucosylated thymine DNA base (␤-D-glucosyl-hydroxymethyluracil or base J) is present within sequences flanking the polycistronic units (PTUs) in T. cruzi. The loss of base J at sites of transcription initiation, via deletion of the two enzymes that regulate base J synthesis (JBP1 and JBP2), correlates with an increased rate of Pol II transcription and subsequent genome-wide increase in gene expression. The affected genes include virulence genes, and the resulting parasites are defective in host cell invasion and egress. These studies indicate that base J is an epigenetic factor regulating Pol II transcription initiation in kinetoplastids and provides the first biological role of the only hypermodified DNA base in eukaryotes.Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is the major cause of cardiac disease in South and Central America (1). The parasite has a complex life cycle with two hosts and four developmental stages. Epimastigotes develop in the hindgut of the triatomine insect vector and differentiate into metacyclic forms. Infective metacyclic forms enter the vertebrate host, invade the host cell, and differentiate to form amastigotes. Trypomastigotes released from the infected cell are able to reinvade a wide variety of host cells. Success of the parasite throughout the life cycle is ensured by the regulated expression of surface proteins such as mucin and trans-sialidase, which allow differential adherence and evasion of the host immune responses (2). Members of the surface glycoprotein gene family colocalize with a novel hypermodified DNA base, ␤-D-glucosyl-hydroxymethyluracil or base J, suggesting an epigenetic mechanism of regulating T. cruzi pathogenesis (3).In base J, the thymine base exhibits O-linked glucosylation in telomeric DNA of all kinetoplastid flagellates and some closely related unicellular flagellates, but base J is not present in the genomes of other protozoa or metazoa (3, 4). Base J was initially discovered on the basis of its distinct presence within the 19 silent telomeric variant surface glycoprotein (VSG) expression sites (ES) of Trypanosoma brucei but absence from the single transcribed ES, suggesting its role in the regulation of telomeric VSG gene expression (3, 35). Recent genomewide analysis revealed that base J is also present throughout the T. brucei genome, enriched at regions flanking polymerase II (Pol II) polycistronic transcription units (PTUs) (9). PTUs are large gene clusters that are cotranscribed by Pol II to yield polycistronic pre-mRNAs that are then processed into mature mRNAs by trans-splicing and polyadenylation (7). The localization of base J at PTU-flanking regions suggests a role for the modified base in regulating Pol II transcription initi...

, Fig 1E: The tubulin control hybridization was a misplaced one. Since we are unable to locate the correct tubulin control hybridization that goes with the original figure, another anti-base J blot and corresponding tubulin hybridization were produced. The DNA dot blot was first probed with anti-J to measure base J levels, and then the same blot was hybridized with the tubulin probe. The hybridizations were then spliced in order to present the samples that correspond to the indicated cell lines. The corrected figure is shown below. The conclusion remains unchanged by this correction. We apologize for any confusion this error might have caused.