BMC Genomics 2008, 9:298 http://www.biomedcentral.com/1471-2164/9/298 while Tb927.7.6440 and Tb927.3.3720 do not display significant similarity to any specific SNARE. By contrast, Tb10.61.0870, is the only upregulated SNARE message in the procyclic stage, with weak similarity to Aspergillus Bos1, suggesting a putative role in anterior transport from the ER; Tb10.61.0870 is also the most highly expressed Qb SNARE (Figure 3). These data are suggestive of specific upregulation of mRNAs encoding endocytic and exocytic SNAREs in BSF, with evidence for limited PCF regulation of the secretory pathway. On average, Qc and R SNAREs are more highly expressed than Qa and Qb SNAREs (Fig- ure 3). The T. brucei N-ethylmaleimide sensitive factor (NSF, Tb927.1.1560), an ATPase responsible for regula- tion of SNARE complex disassembly [60], and a hypothet- ical protein similar to γ-soluble NSF attachment protein (γ-SNAP, Tb10.70.4620) are upregulated in BSF, which may indicate increased overall SNARE activity, but a fur- ther putative SNAP (Tb927.1.3110) is upregulated in PCF. Interestingly, both Tb927.1.1560 (NSF) and Tb927.1.3110 (SNAP) have severe RNAi knockdown growth phenotypes [61]. Expression of Vps16, a compo- nent of the HOPS tethering complex functioning at the late endosome, is increased in the BSF (Table 1). Overall, multiple factors likely to be controlling endocytic path- ways, encompassing SNAREs, Rabs and possibly also the HOPS complex, appear more highly expressed in the BSF, consistent with earlier data but also suggesting more extensive remodelling than previously suspected. Coat proteins The vast majority of vesicle coat factor mRNAs are expressed at equivalent levels in both major life stages. Of those that are regulated, most notable is the clathrin light chain, Tb10.61.1910, upregulated in BSF and in agree- ment with increased endocytosis in this life stage [16]. Further, Tb11.01.6880, containing a predicted emp24 domain, suggesting a role as a cargo receptor in COPII- mediated transport [62], is upregulated in BSF, whereas a second emp24-related ORF, Tb927.4.4350, is upregulated in PCF. Should these proteins indeed represent emp24 isoforms, this expression profile indicates potential devel- opmental-stage specific remodelling of COPII cargo selec- tion providing differentiation in ER exit pathways. A putative epsinR (Tb11.50.0006), is also significantly upregulated in BSF; epsinR is associated with clathrin coated vesicles and AP1 [63]. The AP1 subunit is signifi- cantly upregulated in BSF, but the μ-subunit of AP3, is upregulated in PCF (Table 1); both complexes have a role in endosome to TGN transport. Although no further adap- tin components exhibit highly significant differential expression, a general trend of upregulation of AP1 compo- nent in BSFs and downregulation of AP3 and AP4 was evi- dent on the microarray [see Additional file 3] which suggests developmental regulation. From previous analy- ses, the AP1 γ-subunit is constitutively expressed [64], but the β-chain is upregulated in BSF [16], suggesting possible subunit-specific regulation. To further confirm the signif- icance of these data polyclonal antibodies were raised against the δ-subunit of AP3. Western blotting confirmed significant developmental regulation of AP3δ, with expression greatly augmented in the PCF [see Additional file 3]. These data are also consistent with evidence sug- gesting increased emphasis on lysosomal trafficking in the PCF [26,65]. Proteases Several proteases are known components of the trypano- some surface and endocytic system or predicted to be, based on the presence of a signal peptide. The gp63 iso- form TbMSP-B (Tb927.8.1610), is upregulated in PCF, while TbMSP-C (Tb10.70.5290) is upregulated in BSF (Table 1), confirming previous analyses [52]. TbMSP-A, previously identified as BSF-specific [52], is only slightly upregulated by microarray, but is validated strongly by qRT-PCR (results not shown). Further, metacaspase MCA3 (Tb927.6.930) showed significant upregulation in BSF, also confirming earlier data [66]. Microarray data indicate that MCA5 (Tb09.211.4760) is upregulated in PCF, but qRT-PCR data failed to validate this, suggesting more equivalent expression, also in agreement with ear- lier work [66]. Two subtilysin-like serine peptidases (Tb927.3.4230, Tb11.02.1280), a CAAX prenyl protease (Tb09.211.0680), and a thimet oligopeptidase A (Tb927.7.190) are upregulated in BSF. The thimet oli- gopeptidase Tb927.7.190 was recently suggested to play a role in dysregulated kinin metabolism observed in the plasma of trypanosome-infected hosts and to contribute to vascular lesions observed in African trypanosomiasis [67], consistent with augmented expression in BSF. The most abundant protease class message is for Tb927.6.960, an L-cathepsin-like cysteine peptidase, and likely a lysosomal protein [68], which appears equiva- lently expressed in both life stages (Figure 3). Some of the developmentally regulated proteases are also abundant at the mRNA level (Figure 3), namely MSP-B (Tb927.8.1610), MCA5 (Tb09.211.4760), a subtilysin- like serine peptidase (Tb927.3.4230), and thimet oli- gopeptidase A (Tb927.7.190). Hence the major proteo- lytic activity likely resides within the cysteine protease class, as previously reported [69], but these new data sug- gest a significant level of developmental remodelling, as well as potential contributions from additional protease classes. Chaperones and ER lumenal proteins Several putative ER chaperones, likely to aid folding of secreted coat proteins, were also studied. Of these, Tb927.7.5790 is homologous to ERp44, a thioredoxin of Page 10 of 28 (page number not for citation purposes)