2768 Research Article Introduction The primary organelles of the secretory pathway are the endoplasmic reticulum (ER) (Porter et al., 1945) and the Golgi complex (Golgi, 1898). The trafficking of membranes between major elements of the secretory pathway is largely mediated by discontinuous carriers (Palade, 1975). The specificity of trafficking pathways depends on the selective incorporation of cargo into newly forming transport intermediates, movement of these transport intermediates to the target membrane and subsequent recognition and fusion steps (Palade, 1975). Virtually all of these steps are regulated by small Ras-family proteins in the Arf/Arl (Donaldson et al., 2005; Kahn et al., 2006) and Rab (Stenmark and Olkkonen, 2001; Zerial and McBride, 2001) subfamilies. Many Rabs are known to regulate distinct aspects of trafficking pathways, often by regulating tethers, SNARE (soluble NSF attachment receptor) proteins and motors (Zerial and McBride, 2001). For example, Rab5 binds to early endosome antigen 1 (EEA1) and forms a complex with SNARE proteins to mediate homotypic fusion of early endosomes (Christoforidis et al., 1999; McBride et al., 1999). Rab1 interacts with other tethers including p115 required for the fusion of pre-Golgi intermediates with Golgi membranes (Sztul and Lupashin, 2006). Rab6 regulates a microtubule/kinesin-dependent pathway originating at the trans-Golgi network (TGN) (Echard et al., 1998; Girod et al., 1999; White et al., 1999). Thus, many Rab proteins possess a characteristic organellar distribution and regulate a specific transport step (Chavrier et al., 1990; Zerial and McBride, 2001). There are in excess of 60 Rab-family (Pereira-Leal and Seabra, 2001) and 29 Arf/Arl (Kahn et al., 2006) proteins in humans compared with 11 Rab/Ypt proteins (Lazar et al., 1997) and five Arf/Arls in yeast (Behnia et al., 2004; Huang et al., 1999). Studies in mammalian cells have provided evidence for organization of specific Rabs in ‘Rab domains’ (Sonnichsen et al., 2000), which possess distinct lipid compositions but may be defined in part also by Rab effector proteins capable of interacting simultaneously with different Rabs (De Renzis et al., 2002). Studies in yeast also find recruitment of Rabs to lipid domains and it has been proposed based on these data that lipids define in part the localization of Rab proteins (Sciorra et al., 2005). Arfs are found in pits coated with COPI (coat protein complex) or clathrin which may indicate the existence of other spatially delimited domains. Because of their central roles, Arf/Arl and Rab proteins provide appealing initial starting points for molecular characterization of these trafficking pathways. The increased number of these proteins in mammals is believed primarily to derive from the larger number of trafficking pathways, particularly between the Golgi complex, cell surface and endosomes, in addition to the variety of cargo requiring transport. As a consequence, in more complex organisms, many Rab/Arf/Arl proteins would be expected to associate with the different Rabs and Arfs/Arls are Ras-related small GTPases of particular relevance to membrane trafficking. It is thought that these proteins regulate specific pathways through interactions with coat, motor, tether and SNARE proteins. We screened a comprehensive list of Arf/Arl/Rab proteins, previously identified on purified Golgi membranes by a proteomics approach (37 in total), for Golgi or intra-Golgi localization, dominant-negative and overexpression phenotypes. Further analysis of two of these proteins, Rab18 and Rab43, strongly indicated roles in ER-Golgi trafficking. Rab43-T32N redistributed Golgi elements to ER exit sites without blocking trafficking of the secretory marker VSVG-GFP from ER to cell surface. Wild-type Rab43 redistributes the p150 Glued subunit of dynactin, consistent with a specific role in regulating association of pre-Golgi intermediates with microtubules. Overexpression of wild-type GFP-Rab18 or incubation with any of three siRNAs directed against Rab18 severely disrupts the Golgi complex and reduces secretion of VSVG. Rab18 mutants specifically enhance retrograde Golgi-ER transport of the COPI-independent cargo β-1,4-galactosyltransferase (Galtase)-YFP but not the COPI- dependent cargo p58-YFP from the Golgi to ER in a photobleach assay. Rab18-S22N also potentiated brefeldin-A-induced ER- Golgi fusion. This study is the first comprehensive application of large-scale proteomics to the cell biology of small GTPases of the secretory pathway. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/121/16/2768/DC1 Key words: ER-Golgi Trafficking, GFP, Golgi, Rab18, Rab43, Endoplasmic reticulum Summary Rab18 and Rab43 have key roles in ER-Golgi trafficking Selma Y. Dejgaard 1 , Ayesha Murshid 1 , Ays ¸egül Erman 1 , Özge Kızılay 1 , David Verbich 1 , Robert Lodge 2 , Kurt Dejgaard 3 , Thi Bach Nga Ly-Hartig 4 , Rainer Pepperkok 4 , Jeremy C. Simpson 4, * and John F. Presley 1,‡ 1 Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada, H3A 2B2 2 Laboratoire d’Immunoretrovirologie, Centre de Recherche d’Infectiologie – CHUL, Quebec, Canada, G1V 4G2 3 Department of Biochemistry, McGill University, Montreal, Quebec, Canada, H3G 1Y6 4 Cell Biology and Biophysics Unit, EMBL, 69117 Heidelberg, Germany *Present address: School of Biology and Environmental Science, UCD, Dublin, Ireland ‡ Author for correspondence (e-mail: john.presley@mcgill.ca) Accepted 19 May 2008 J. Cell Sci. 121, 2768-2781 Published by The Company of Biologists 2008 doi:10.1242/jcs.021808 Journal of Cell Science