Malaria parasites secrete proteins across the vacuolar membrane into the erythrocyte, inducing modifications linked to disease and parasite survival. We identified an 11-amino acid signal required for the secretion of proteins from the Plasmodium falciparum vacuole to the human erythrocyte. Bioinformatics predicted a secretome of >320 proteins and conservation of the signal across parasite species. Functional studies indicated the predictive value of the signal and its role in targeting virulence proteins to the erythrocyte and implicated its recognition by a receptor/transporter. Erythrocyte modification by the parasite may involve plasmodial heat shock proteins and be vastly more complex than hitherto realized.

When the malaria parasite Plasmodium falciparum infects an erythrocyte, it resides in a parasitophorous vacuole and remarkably exports proteins into the periphery of its host cell. Two of these proteins, the histidine-rich proteins I and II (PfHRPI and PfHRPII), are exported to the erythrocyte cytoplasm. PfHRPI has been linked to cell-surface ''knobby'' protrusions that mediate cerebral malaria and are a frequent cause of death. PfHRPII has been implicated in (i) the production of hemozoin, the black pigment associated with disease, as well as (ii) interactions with the erythrocyte cytoskeleton. Here we show that a tripartite signal that is comprised of an endoplasmic reticulum-type signal sequence followed by a bipartite vacuolar translocation signal derived from HRPII and HRPI exports GFP from the parasitophorous vacuole to the host cytoplasm. The bipartite vacuolar translocation signal is comprised of unique, peptidic (Ϸ40-aa) sequences. A domain within it contains the signal for export to ''cleft'' transport intermediates in the host erythrocyte and may thereby regulate the pathway of export to the host cytoplasm. A signal for posttranslational, vacuolar exit of proteins has hitherto not been described in eukaryotic secretion.

Polar replication fork barriers (RFBs) near the 3 end of the rRNA transcriptional unit are a conserved feature of ribosomal DNA (rDNA) replication in eukaryotes. In the mouse, in vivo studies indicate that the cis-acting Sal boxes required for rRNA transcription termination are also involved in replication fork blockage. On the contrary, in the budding yeast Saccharomyces cerevisiae, the rRNA transcription termination factors are not required for RFBs. Here we characterized the rDNA RFBs in the fission yeast Schizosaccharomyces pombe. S. pombe rDNA contains three closely spaced polar replication barriers named RFB1, RFB2, and RFB3 in the 3 to 5 order. The transcription termination protein reb1 and its two binding sites, present at the 3 end of the coding region, were required for fork arrest at RFB2 and RFB3 in vivo. On the other hand, fork arrest at the strongest RFB1 barrier was independent of the above transcription termination factors. Therefore, RFB2 and RFB3 resemble the barriers present in the mouse rDNA, whereas RFB1 is similar to the budding yeast RFBs. These results suggest that during evolution, cis-and trans-acting factors required for rRNA transcription termination became involved in replication fork blockage also. S. pombe is suggested to be a transitional species in which both mechanisms coexist.During eukaryotic ribosomal DNA (rDNA) replication, the fork moving opposite to transcription is arrested at replication fork barriers (RFBs) close to the 3Ј end of the coding region (3, 13, 24-27, 42, 43). RFBs must play a relevant biological role, since they are highly conserved in eukaryotes. Due to the polar nature of RFBs, rDNA is replicated mainly in a unidirectional mode cooriented with transcription. Thus, one possible role for the RFB may be to prevent the deleterious effects of head-on collisions between replication and transcription machineries (32). Since the DNA sequence at the RFB is not sufficient per se to stall replication (4, 28), fork arrest must be induced by a protein factor(s) bound to the rDNA at the barrier.In Saccharomyces cerevisiae rDNA, protein Fob1 is required for RFB activity (19), although it is still unknown whether it arrests rDNA replication by binding to the RFB sites or through a different mechanism. Functional RFBs are required for HOT1 recombination, contraction and expansion of the rDNA repeat number, and the formation of extrachromosomal ribosomal circles (17-20), suggesting that RFB activity stimulates recombination occurring at the rDNA locus in this budding yeast (2). On the other hand, it has been recently shown that RFBs and HOT1 recombination are independent activities although they share cis-acting sequences (41).In mouse rDNA, replication forks stall at the rRNA transcriptional terminator elements known as Sal boxes (27), which are the specific binding sites for transcription termination factor mTTF-1 (12, 21). This protein was able to arrest replication forks in an in vitro replication assay (10, 35). These in vivo and in vitro results suggest that Sal boxes...

We analyzed three chromosomal loci of the trypanosomatid Leptomonas collosoma encoding box C/D small nucleolar RNAs (snoRNAs). All the snoRNAs that were analyzed here carry two sequences complementary to rRNA target sites and obey the ؉5 rule for guide methylation. Studies on transgenic parasites carrying the snoRNA-2 gene in the episomal expression vector (pXneo) indicated that no promoter activity was found immediately adjacent to this gene. Deleting the flanking sequences of snoRNA-2 affected the expression; in the absence of the 3-flanking (but not 5-flanking) sequence, the expression was almost completely abolished. The snoRNA genes are transcribed as polycistronic RNA. All snoRNAs can be folded into a common stem-loop structure, which may play a role in processing the polycistronic transcript. snoRNA B2, a member of a snoRNA cluster, was expressed when cloned into the episomal vector, suggesting that each gene within a cluster is individually processed. Studies with permeable cells indicated that snoRNA gene transcription was relatively sensitive to ␣-amanitin, thus supporting transcription by RNA polymerase II. We propose that snoRNA gene expression, similar to protein-coding genes in this family, is regulated at the processing level.