TelAP2 links TelAP1 to the telomere complex in Trypanosoma brucei

Abstract

The extracellular parasite Trypanosoma brucei evades the immune system of the mammalian host by periodically exchanging its variant surface glycoprotein (VSG) coat. Hereby, only one VSG gene is transcribed from one of 15 subtelomeric so-called bloodstream form expression sites (BES) at any given timepoint, while all other BESs are silenced. VSG gene expression is altered by homologous recombination using a large VSG gene repertoire or by a so-called in situ switch, which activates a previously silent BES. Transcriptional activation, VSG switching and VSG silencing during developmental differentiation from the bloodstream form to the procyclic form present in the tsetse fly vector are tightly regulated. Due to their subtelomeric position, telomere-associated proteins are involved in the regulation of VSG expression. Three functional homologs of mammalian telomere complex proteins have been characterized thus far, and novel telomere-interacting proteins, such as telomere-associated protein 1 (TelAP1), have recently been identified. Here, we used mass spectrometry-based proteomics and interactomics approaches, telomere pull-down assays with recombinant material and immunofluorescence analysis to elucidate the interactions of 21 other putative TelAPs. We investigated the influence on VSG expression and showed that depletion of TelAPs does not ultimately lead to changes in VSG expression. Additionally, we examined the interaction patterns of four TelAPs with the TbTRF/TbTIF2/TbRAP1 telomere complex by reciprocal affinity purification. We further propose that TelAP1 interacts with Tb927.6.4330, now called TelAP2, and that TelAP1 depends on this interaction to form a complex with the telomeric proteins TbTRF, TbTIF2 and TbRAP1.

Keywords: Trypanosoma brucei; Parasitology; Telomere complex.

Fig. 1

VSG expression pattern after depletion of telomere-associated protein candidates. (A) Enrichment of previously silent VSGs in induced (+) compared to uninduced (-) and wild-type (WT) cells is displayed as a percentage of the total VSG. VSG-2 (92–99% abundance) was excluded from all the graphs. RNAi was induced by adding tetracycline (tet). Cell lines that exhibited a growth phenotype after protein depletion are underlined. VSG proteins were released from the cell surface and analyzed by mass spectrometry. The published VSG expression patterns of TbRAP1- and PolIE-depleted cells were added for comparison. (B) Close-up of the expression patterns of previously silenced BES (left panel), MES (middle panel) and internal VSGs (right panel) of TelAP2, TbRAP1 and PolIE.

Fig. 2

Interactome of telomeric complex proteins. (A–D) Volcano plots generated from the IP-MS data visualizing proteins copurified with the respective PTP-tagged candidates: (A) TelAP2:PTP, (B) PTP:PPL2, (C) TelAP3:PTP and (D) PTP:PolIE. The x-axis shows the log2 enrichment of quantified proteins against the control (either WT or ectopically expressed PTP), and the y-axis represents the p value (Welch t test) of replicate IPs (n = 4). (E) Summary of the interactions detected in the IP experiments among selected telomeric proteins.

Fig. 3

The interaction of TelAP1 with the telomere complex changes upon TelAP2 depletion. IPs were analyzed in quadruplicate by mass spectrometry using a monoclonal TelAP1 mouse antibody, and the results are displayed as a volcano plot. The x-axis shows the enrichment of quantified proteins between the uninduced cell line (control) and the cell line treated with RNAi for (A) TelAP2 or (B) TelAP3; the y-axis represents the p value (Welch t test) of replicates (n = 4). (C) IP results of TbTRF-Ty1 in TelAP2-depleted versus control cell lysates.

Fig. 4

TelAP2 does not bind telomeric DNA in vitro. (A) Volcano plot of TelAP2 IP with and without DNase I treatment. In BSF, none of the known telomeric TelAP2 interaction partners dissociated after treatment. The x-axis shows the enrichment of proteins detected in the untreated lysates compared to the lysates treated with DNase I, and the y-axis represents the p value (Welch t test) of the quadruplicate samples. (B) In vitro telomere-binding assay with recombinant TbTRF and TelAP2. Five micrograms of either purified His-MBP-TbTRF or His-MBP-TelAP2 or a combination of TbTRF and TelAP2 was used in a DNA pulldown assay. The recombinant proteins were incubated with either telomeric (TTAGGG)_n oligonucleotides or control (GTGATG)_n oligonucleotides. (M) Protein size marker.

Fig. 5

Yeast 2-hybrid assay of telomere-associated proteins shows direct interaction between TelAP1 and TelAP2. Interactions were tested in all combinations. For PPL2 and PolIE, the open reading frames were split into two fragments (F1 and F2). Yeast cells transformed with empty prey and bait vectors served as controls. Synthetic complete medium lacking leucine and tryptophane (SC-LW) ensured maintenance of the two plasmids. Growth in the absence of histdine (SC-LWH) versus histidine and adenine (SC-LWHA) indicates interactions under low versus high stringency.