BMC Genomics 2008, 9:298 http://www.biomedcentral.com/1471-2164/9/298 the protein disulfide isomerase (PDI) family, which may have a role in controlling the redox state of the ER, and is upregulated in BSF [70]. Two additional PDI proteins are also upregulated in the bloodstream stage of T. brucei [71,72], suggesting remodelling of these factors between life stages. In the PCF stage, two predicted mitochondrial chaper- ones, an Hsp70 (Tb927.6.3740) and an Hsp60 (Tb10.70.0280), are upregulated, consistent both with previous data [73,74] and increased mitochondrial activ- ity [6]. Additionally, an Hsp70 (Tb11.01.3110) with high similarity to human Hsc70, an uncoating ATPase for clathrin coated vesicles, is upregulated in PCF [75-77]. Since clathrin-mediated endocytosis is upregulated in BSF but lysosomal targeting upregulated in PCF, these data may indicate a role in lysosomal targeting, but there are multiple Hsp70 ORFs in T. brucei and Hsp70 has been previously reported to be upregulated slightly in BSF [78]. Two novel ER lumenal proteins, Tb11.01.2640 and Tb11.01.8120, with a predicted signal peptide and C-ter- minal HDEL ER-retrieval motif are developmentally regu- lated, the former with augmented expression in BSF and the latter in PCF. Both are localised to the ER, and give a severe growth phenotype by RNAi knockdown, suggesting an important function within the ER (T. Sergeenko and MCF, unpublished data). Overall, the expression profile of the chaperones does not provide evidence for substan- tial differential remodelling of the ER folding environ- ment, and may suggest a comparatively similar set of factors are required for synthesis of the cell surface in PCF and BSF, despite major differences in the structures of the superabundant surface antigens. Lipid kinases Two highly conserved lipid kinases with roles in intracel- lular trafficking are known in trypanosomes: Fab1 (Tb11.47.0002), a PI 5-kinase and TbVps34 (Tb927.8.6210), a PI 3-kinase involved in endocytosis and Golgi segregation [35,46]. Neither of these factors shows evidence for differential expression, but several other predicted phosphatidylinositol kinases are develop- mentally regulated (Table 1). A type III phosphatidyli- nositol 4-kinase, Tb927.3.4020, highly similar to S. cerevisiae Stt4p, is upregulated in PCF, consistent with a recent report of a role for a type III PI 4-kinase in protein trafficking, maintenance of Golgi and cell morphology and cytokinesis in PCFs [79]. A predicted phosphatidyli- nositol 3-kinase, Tb10.6k15.2060, is also upregulated in PCF and most likely represents an atypical protein kinase of the TOR-related family [35]. Significantly, human FRAP1, a member of the TOR-related kinase family, is required for clathrin-mediated endocytosis [18]. Tb11.01.6980, an atypical PI 3-kinase with a PI(3)P-bind- ing FYVE domain and a putative N-terminal trans-mem- brane helix is highly upregulated in BSF; on account of the FYVE domain this protein is expected to localise to the endosomal system [35]. Further, Tb10.70.2440, a pre- dicted PI4P 5-kinase, is also upregulated in BSF, while two serine threonine kinases with pleckstrin homology (PH) domains, capable of recognising PI4,5P lipids, are upreg- ulated in BSF. Taken together these data suggest signifi- cant remodelling of the PI-based signalling system between life stages, together with evidence for increased expression of several putative endosomal functioning PI- kinases in the BSF. Protein kinases A genome-wide analysis of the roles of kinases in clathrin- and caveolin-mediated endocytosis was performed recently in H. sapiens [18]; significantly, caveolin-medi- ated endocytosis is restricted to the metazoa [46]. We looked for orthologues of the kinases that gave an endo- cytosis phenotype in the Pelkmans et al . study [18] and included these in the array to analyse their differential expression. Two metabolic pathway kinases and three putative signal- ling kinases are upregulated in BSF. These include Tb10.70.5800, a glycosomal hexokinase, expected due to increased glycolytic activity in BSFs [14,80]; this kinase is similar to H. sapiens hexokinase II, required for clathrin- mediated endocytosis [18]. Pyruvate kinase (Tb10.61.2680) is also upregulated, in agreement with earlier work [81], and interestingly the related H. sapiens kinase, PKLR, inhibits caveolae-mediated endocytosis [18]. The three signalling kinases upregulated in BSF all show similarities to endocytosis kinases [18]. Tb927.6.1780, a putative MAP-kinase similar to human MAPK8, is activated in response to temperature stress [34]. A MAP-kinase kinase kinase, Tb10.61.2490, has sim- ilarity to human MAP3K8, and finally a calcium-depend- ent protein kinase, Tb927.4.3770, is highly similar to CAMK4, which participates in a calcium/calmodulin- dependent protein kinase cascade [82]. These data clearly suggest a role for kinase activity in BSF trafficking. Eight primarily metabolic pathway kinases are upregu- lated in PCF. One is a nucleoside diphosphate kinase (Tb11.01.7800) which is involved in the equilibration of cellular pools of nucleotide triphosphates, and which binds telomeres and acts as a Rho-GAP in other systems [83-85]. This protein localises predominantly to the nucleus in T. brucei [86] and shows similarity to human NME6, found to inhibit clathrin-mediated endocytosis [18]. A ribokinase (Tb11.03.0090) upregulated in PCF is highly similar to human RBSK, required for clathrin- mediated endocytosis [18]. A putative NEK kinase (Tb927.3.3190) is related to human NEK2, 6, and 7, all of which are required for clathrin-mediated endocytosis Page 11 of 28 (page number not for citation purposes)