1 Introduction Fink, 1999 Hennessy, Nicoll, Zimmermann, Cheetham, & Blatch, 2005b Schmid, Baici, Gehring, & Christen, 1994 Cheetham, Jackson, & Anderton, 1994 Liberek, Marszalek, Ang, Georgopoulos, & Zylicz, 1991 Han & Christen, 2003 Suh, Lu, & Gross, 1999 Auger & Roudier, 1997 Hennessy et al., 2005b; Suh et al., 1999 Cheetham & Caplan, 1998 Langer et al., 1992 Rüdiger, Schneider-Mergener, & Bukau, 2001 Garimella et al., 2006; Hennessy et al., 2005b E. coli Huang, Flanagan, & Prestegard, 1998 Pellecchia, Szyperski, Wall, Georgopoulos, & Wüthrich, 1996 Qian, Patel, Hartl, & McColl, 1996 E. coli Cupp-Vickery & Vickery, 2000 Berjanskii et al., 2000 Kim, Ahn, & Cho, 2001 Gruschus, Greene, Eisenberg, & Ferretti, 2004 Jiang et al., 2003 Genevaux, Wawrzynow, Zylicz, Georgopoulos, & Kelley, 2001 Laufen et al., 1999 Mayer, Laufen, Paal, McCarty, & Bukau, 1999 Tsai & Douglas, 1996 Wittung-Stafshede, Guidry, Horne, & Landry, 2003 Hennessy, Cheetham, Dirr, & Blatch, 2000 Hennessy, Boshoff, & Blatch, 2005a Garimella et al., 2006 Genevaux, Schwager, Georgopoulos, & Kelley, 2002 Genevaux et al., 2003 Lu & Cyr, 1998 Suh et al., 1999 E. coli Hill, Flanagan, & Prestegard, 1995 Pellecchia et al., 1996 Szyperski, Pellecchia, Wall, Georgopoulos, & Wuthrich, 1994 Hennessy et al., 2000 E. coli Hennessy et al., 2005a E. coli Agrobacterium tumefaciens Genevaux et al., 2002; Hennessy et al., 2005a,b Genevaux et al. (2002) E. coli Genevaux et al., 2002 Kelley & Georgopoulos, 1997 Laufen et al., 1999; Mayer et al., 1999; Tsai and Douglas, 1996 Genevaux et al. (2002) Hennessy et al., 2000 E. coli Genevaux et al., 2002 Hennessy et al., 2005a Hennessy et al., 2005a Pellecchia et al., 1996 Genevaux et al., 2002 Hennessy et al., 2005a,b E. coli Suh et al., 1999 Hennessy et al., 2005a Garimella et al., 2006; Hennessy et al., 2005b E. coli Brodsky, Hamamoto, Feldheim, & Schekman, 1993 Wiech, Buchner, Zimmermann, Zimmermann, & Jakob, 1993 E. coli Minami, Höhfeld, Ohtsuka, & Hartl, 1996 Hennessy et al., 2005b Deloche, Kelley, & Georgopoulos, 1997 Genevaux et al., 2001 Schlenstedt, Harris, Risse, Lill, & Silver, 1995 Kelley and Georgopoulos, 1997; Sullivan et al., 2000 In this study we have conducted domain swapping of the J-domains from a selection of divergent Hsp40s of mammalian and parasitic origin (malarial Pfj1 and Pfj4, trypanosomal Tcj3, human ERj3, ERj5, and Hsj1, and murine ERj1) in an attempt to identify system specific and common factors in Hsp40–Hsp70 interactions. The similarities and differences in the structure and function of Hsp40s of parasites and their hosts have yet to be determined, and therefore from this broader perspective we were interested in a comparative analysis of the J-domains of parasitic and human origin. An in vivo functional assay was used to assess the ability of the J-domains to substitute for the J-domain of a prokaryotic type I Hsp40. Furthermore, the functional importance of specific residues was addressed through single amino acid substitution analysis. The data suggested that cytosolic and ER J-domains of mammalian and parasitic origin can interact with DnaK using a common mechanism, and that a fundamental binding surface appears to be conserved in J-domains of Hsp40s of mammalian, parasitic and bacterial origin. 2 Materials and methods 2.1 Materials E. coli araD139 ara714 cbpA kan dnaJ Edkins, Ludewig, & Blatch, 2004 2.2 Creation of the Agt DnaJ chimera proteins Hennessy et al., 2005a Bst Fig. 1 Bst Bam Fig. 1 E. coli Plasmodium falciparum Fig. 1 Nicoll et al., 2006 Stemmer, Crameri, Ha, Brennan, & Heyneker, 1995 Bam Bst Bam Bst 2.3 In vivo complementation assays E. coli dnaJ cbpA Deloche et al., 1997; Kelley and Georgopoulos, 1997 E. coli E. coli Hennessy et al., 2005a E. coli Hennessy et al., 2005a E. coli E. coli 600 600 −8 E. coli 2.4 Western analysis for the detection of chimeras E. coli 6 2.5 Binding studies with ERj1-J and BiP Brightman, Blatch, & Zetter, 1995 Dudek et al., 2002 Tyedmers et al., 2000 2 3 Results 3.1 Bioinformatic analysis of the J-domain and the identification of structurally and functionally important residues Fig. 2 E. coli dnaJ cbpA E. coli Hennessy et al., 2005a E. coli E. coli E. coli Hennessy et al., 2005a E. coli E. coli E. coli E. coli Pröls et al., 2001 Trypanosoma cruzi Edkins et al., 2004 P. falciparum Watanabe, 1997 Nakai & Kanehisa, 1992 Watanabe, 1997 Cheetham, Brion, & Anderton, 1992 Chapple & Cheetham, 2003 Brightman et al., 1995 Dudek et al., 2002, 2005 Chevalier, Rhee, Elguindi, & Blond, 2000 Dudek et al., 2002 Bies et al., 1999, 2004 Yu & Haslam, 2005 Cunnea et al., 2003 Hosoda, Kimata, Tsuru, & Konho, 2003 Cunnea et al., 2003 E. coli Fig. 2 Fig. 2 E. coli Fig. 2 Hennessy et al., 2000 Fig. 2 E. coli Fig. 2 Fig. 2 Genevaux et al., 2002; Hennessy et al., 2005a 3.2 Characterization of the chimeras E. coli E. coli −8 E. coli Fig. 3 E. coli Hennessy et al., 2005a E. coli E. coli Fig. 3 Table 1 E. coli E. coli E. coli Fig. 3 E. coli 3.3 Pfj1, Pfj4 and Hsj1 J-domain mutants E. coli E. coli Table 1 E. coli Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 R R K Table 1 Hennessy et al., 2005a E. coli 3.4 ERj1 J-domain Fig. 4 Fig. 4 Fig. 4 4 Discussion E. coli E. coli Genevaux et al., 2002 Hennessy et al., 2005a,b 4.1 J-domain interchangeability E. coli Hennessy et al., 2005a P. falciparum Matambo, Odunuga, Boshoff, & Blatch, 2004 Sargeant et al., 2006 Garimella et al., 2006; Hennessy et al., 2005a,b E. coli Schlenstedt et al. (1995) E. coli E. coli Pröls et al., 2001 Kluck et al. (2002) E. coli E. coli E. coli E. coli Kluck et al., 2002 Chevalier et al., 2000; Dudek et al., 2002 E. coli E. coli Chevalier et al., 2000 4.2 Targeted mutagenesis 4.2.1 Helix I Berjanskii et al., 2000 Cupp-Vickery & Vickery, 1997, 2000 Huang et al., 1998; Pellecchia et al., 1996; Qian et al., 1996 Berjanskii et al., 2000 Hennessy et al. (2005a,b) Hennessy et al., 2005a 4.2.2 Helix II and the loop Berjanskii et al., 2000 E. coli Genevaux et al., 2002; Hennessy et al., 2005a,b E .coli Genevaux et al. (2002) E. coli Genevaux et al., 2002 E. coli 4.2.3 Helix III Genevaux et al., 2002 Genevaux et al., 2002 Hennessy et al., 2000; Landry, 2003 E. coli E. coli Genevaux et al., 2002; Hennessy et al., 2005a,b Johnson & Craig, 2000 Hennessy et al., 2005a,b Hennessy et al., 2005a,b Hennessy et al., 2005a,b 4.2.4 Helix IV Hennessy et al. (2005a,b) Genevaux et al. (2002) E. coli E. coli Genevaux et al., 2002 Garimella et al., 2006; Hennessy et al., 2005a,b 4.3 Conclusion and future perspectives This study has analysed cytosolic and ER Hsp40s of mammalian and parasitic origin, and found that certain key features of the J-domain appear to be fundamental to the function of all the J-domains studied, and perhaps to the function of J-domains in general. Interestingly, the differences appear to be subtle (e.g. the effects of the Y7A and R26A substitutions on the function of the J-domains of Hsj1 versus Pfj4), and may reflect slight differences in affinity or specificity of these J-domains for DnaK. These differences need to be probed further using quantitative in vitro assays, and incorporating an analysis of the less-conserved J-domain residues shown by NMR analysis to occur at J-domain-DnaK/Hsp70 binding interfaces. Furthermore, the possibility that there are specialized features unique to the J-domains of integral-membrane-bound Hsp40s needs to be investigated more extensively.