sembly of an actin mesh surrounding an organelle can pro- vide active zones where vesicles can fuse or bud (Lorra and Huttner, 1999). It is plausible that Rho1p acts as a spatial landmark to start an actin remodeling event leading to mem- brane fusion (Eitzen, 2003). Recently, a systems approach to study regulators of yeast peroxisome biogenesis identiﬁed signaling proteins involved in actin organization and/or phosphatidylinositol metabolism (Saleem et al., 2008). Many of these proteins function through activation by Rho1p. To control actin dynamics, Rho GTPases either target regulators of actin nucleation, or change the abundance of membrane phosphoinositides to affect the recruitment of actin-binding proteins (Ridley, 2006). Interestingly, lipid raft-like mem- brane domains enriched with ergosterol and ceramide have been found on peroxisome precursors that act as signaling platforms to organize the fusion machinery including phos- phoinositides and small GTPases (Boukh-Viner et al., 2005). Alternatively, Rho1p-dependent actin dynamics are used to change membrane tension and, in the presence of phosphoi- nositide-binding proteins that can assemble into a scaffold at discrete sites, can generate membrane curvature necessary for vesicle budding/fusion (McMahon and Gallop, 2005). All members of the Pex11-family proteins can interact with themselves, and some with each other, suggesting the ability to oligomerize (Thoms and Erdmann, 2005). Evidence also points to Pex-11 family proteins being able to bind phospho- lipids and playing a direct role in membrane modiﬁcation (Fagarasanu et al., 2007). This would be consistent with a previous proposal that Rho1p might facilitate Pex11p activ- ity based on the actin phenotype observed for a pex25 pex11 double mutant (Marelli et al., 2004). The fact that SifA can affect Rho1p activity and change yeast peroxisome morphology and distribution is reminis- cent of its role on the assembly and tubulation of the SCV during Salmonella infection. SifA effects on membrane dy- namics can be drawn in parallel between yeast and mam- malian cells. In both systems, SifA is involved in exagger- ated membrane tubulation (Sif- and SifA-induced tubules in epithelial cells or invaginated peroxisomes in yeast); in con- junction with a lipid-modifying protein (Salmonella effector SseJ; or possibly Pex25p) and the small GTPase Rho-family proteins (Brumell et al., 2002; Ruiz-Albert et al., 2002; Ohlson et al., 2008). SseJ, like Pex25p, recruits RhoA (the mammalian equivalent of Rho1p) and in turn, is activated by RhoA to alter the membrane composition of cholesterol, a condition that can induce endosomal tubules (Christen et al., 2009). SifA can bind RhoA (Ohlson et al., 2008) and might recruit Rho proteins directly to both membrane systems. This can amplify RhoA effects on SseJ or other bacterial or host lipid- modifying proteins, resulting in SifA-induced phagosome tubulation, or can overpromote Rho1p effects on peroxi- somes causing aberrant membrane invaginations. Consis- tent with this model, the ability of SifA to induce tubules in combination with SseJ was reduced in the presence of siRNA against Rho proteins (Ohlson et al., 2008), and SifA does not reduce the number of free peroxisomes in a rho1 or pex25 mutant. Interestingly, peroxisomal membrane in- vaginations are reminiscent of the phenotype in the lipase mutant lpx1 with defects in membrane lipid modiﬁcation (Thoms et al., 2008). It is possible that SifA expression dis- rupts Rho1p normal regulation of a yeast lipase by promot- ing its membrane modifying activity at an inappropriate time during peroxisome biogenesis, causing an imbalance between membrane growth and ﬁssion and hence, the ex- aggerated invagination phenotype. A useful consequence of studying SifA activity on yeast peroxisome division is that this could be a means to increase Rho1p activity speciﬁcally Vol. 21, October 15, 2010 Salmonella SifA on Yeast Peroxisomes on peroxisomes without having to overexpress Rho1p, a condition that could be lethal to vegetative growth. Yeast Peroxisomes as Model System to Elucidate SifA Function Inferring from the yeast studies, we reexamined the distri- bution of SifA in mammalian cells and found that GFP-SifA did not speciﬁcally colocalize with the peroxisomal protein PMP70 in most cells. There have been previous examples of bacterial virulence proteins that target common structural components of eukaryotic signaling pathways (e.g., MAP kinase) even though the outcomes of these pathways may not necessarily be conserved between mammalian and yeast systems (e.g., inﬂammation vs. mating; Valdivia 2004; Sig- gers and Lesser 2008). Because GFP-SifA still localizes to peroxisomes in pex25 mutants, it is plausible that a com- mon protein exists on both yeast peroxisomes and mamma- lian endosomes that recruits SifA to the relevant membrane systems and link it to the local conserved Rho family mem- bers. This membrane recruiter would also have different cytoskeletal interactions. For example, there is a pronounced abundance of microtubules in mammalian cells compared with yeast cells, and there is also no homologue for SKIP in yeast cells that could link SifA to microtubules. Because many aspects of endosomal fusion leading to SCV assembly are unknown, the studies described here could provide a model system to help deﬁne additional binding partners for SifA and its effects on Rho proteins and mem- brane dynamics. Speciﬁcally, the recruitment and activation of a GTPase at an inappropriate time in the functional de- velopment of the phagosome could result in aberrant mem- brane tubulation. How this activity promotes and maintains the bacteria replication niche remains to be deﬁned. In con- clusion, our data suggest that there are parallels between the effects of a bacterial virulence protein on yeast peroxisomes and the mammalian phagosome that may be exploited to better deﬁne activities of SifA. ACKNOWLEDGMENTS We thank T. Davis (University of Washington), D. Drubin (University of California, Berkeley), S. Fields (University of Washington), G. Eitzen (Univer- sity of Alberta, Canada), T. Hazbun (Purdue University), S. Meresse, A. Merz, E. O’Shea, M. Ohl, T. Stevens (University of Oregon, Eugene), Y. Sun (Uni- versity of California, Berkeley), and M. Vignali (University of Washington) for gifts of plasmids and antibodies; T. Davis for usage of the ultra microcentri- fuge; C. Toret for work on GFP-SifA tracking; G. Martin from the Keck Center for advice with microscopy techniques; and M. Brittnacher for help on statis- tics. For discussions, we thank A. Merz; T. Davis, D. Drubin, M. Ohlson, Megha, and A. Haraga, and also thank other members of the Miller lab for their support. Finally, we are grateful to M. Welch and N. Salama for helpful comments on the manuscript. This work was supported by the National Institutes of Health Grant RO1-AI048683 to S.I.M. REFERENCES Abrahams, G. L., Muller, P., and Hensel, M. (2006). Functional dissection of SseF, a type III effector protein involved in positioning the Salmonella-contain- ing vacuole. Trafﬁc 7, 950–965. Alto, N. M., et al. (2006). Identiﬁcation of a bacterial type III effectors family with G protein mimicry functions. Cell 124, 133–145. Beuzon, C. R., Meresse, S., Unsworth, K. E., Ruiz-Albert, J., Garvis, S., Wa- terman, S. R., Ryder, T. A., Boucrot, E., and Holden, D. W. (2000). Salmonella maintains the integrity of its intracellular vacuole through the action of SifA. EMBO J. 19, 3235–3249. Boucrot, E., Beuzon, C. R., Holden, D. W., Gorvel, J. P., and Meresse, S. (2003). Salmonella typhimurium SifA effector protein requires its membrane-anchoring C-terminal hexapeptide for its biological function. J. Biol. Chem. 278(16), 14196–14202. 3575