Molecular Biology of the Cell Vol. 21, 3567–3577, October 15, 2010 Expression of the Salmonella Spp. Virulence Factor SifA in Yeast Alters Rho1 Activity on Peroxisomes Dani B. N. Vinh,* Dennis C. Ko,† Richard A. Rachubinski,‡ John D. Aitchison,§ and Samuel I. Miller*† ¶ Departments of *Medicine, †Immunology, Microbiology, and ¶Genome Sciences, University of Washington, Seattle, WA 98195; ‡Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada; and §Institute for Systems Biology, Seattle, WA 98103 Submitted June 3, 2010; Revised August 2, 2010; Accepted August 17, 2010 Monitoring Editor: Suresh Subramani The Salmonella typhimurium effector protein SifA regulates the assembly and tubulation of the Salmonella phagosome. SifA localizes to the phagosome and interacts with the membrane via its prenylated tail. SifA is a structural homologue of another bacterial effector that acts as a GTP-exchange factor for Rho family GTPases and can bind GDP-RhoA. When coexpressed with a bacterial lipase that is activated by RhoA, SifA can induce tubulation of mammalian endosomes. In an effort to develop a genetic system to study SifA function, we expressed SifA and characterized its activity in yeast. GFP-SifA predominantly localized to yeast peroxisomal membranes. Under peroxisome-inducing conditions, GFP-SifA reduced the number of free peroxisomes and promoted the formation of large peroxisomes with membrane invaginations. GFP-SifA activity depended on the recruitment to peroxisomes of wild-type Rho1p and Pex25p, a receptor for Rho1p. GFP-SifA could also rescue the actin organization defects in pex25 and rho1 mutants, suggesting that SifA may recruit and potentiate Rho1p activity. We reexamined the distribution of GFP-SifA in mammalian cells and found the majority colocalizing with LAMP1-positive compartment and not with the peroxisomal marker PMP70. Together, these data suggest that SifA may use a similar mode of action via Rho proteins to alter yeast peroxisomal and mammalian endosomal membranes. Further deﬁnition of SifA activity on yeast peroxisomes could provide more insight into its role in regulating host membrane dynamics and small GTPases. INTRODUCTION Salmonella are Gram-negative intracellular bacteria that cause diseases ranging from gastroenteritis to typhoid fever (Haraga et al., 2008). The well-studied Salmonella serovar Typhimurium uses type III secretion systems (TTSS), which are essential for pathogenesis and tissue colonization, to translocate SPI (Salmonella pathogenicity island)-1 and -2 effector proteins into the host cytosol. Although there ap- pears to be some overlapping functions, SPI-1 effectors are mainly required during early steps of invading nonphago- cytic cells, and SPI-2 effectors are translocated once bacteria have been internalized. SPI-2 effectors are important for the development of a modiﬁed phagosome called the Salmonella- containing vacuole (SCV) that is required for bacterial sur- vival and replication (Haraga et al., 2008; Steele-Mortimer, 2008). In epithelial cells and macrophages, morphological alterations termed Salmonella-induced ﬁlaments (Sifs) are This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E10 – 06 – 0482) on August 25, 2010. Address correspondence to: Samuel I. Miller (millersi@u. washington.edu). © 2010 D.B.N. Vinh et al. This article is distributed by The American Society for Cell Biology under license from the au- thor(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/li- censes/by-nc-sa/3.0). observed as microtubule-dependent growth extensions of the SCV (Portillo et al., 1993). SCV biogenesis is a dynamic process that results in the progressive accumulation of host endosomal and lysosomal markers. Inside cells, SPI-2 effectors are targeted to the SCV, the Golgi, and the actin cytoskeleton, ultimately promoting alterations of the SCV (Ramsden et al., 2007a; Haraga et al., 2008; Steele-Mortimer, 2008). Microtubule and actin cy- toskeletal ﬁlaments that are rearranged near the SCV and Sifs require speciﬁc SPI-2 effectors (Meresse et al., 2001; Brumell et al., 2002; Miao et al., 2003; Kuhle et al., 2004; Poh et al., 2008). Some SPI-2 effectors manipulate microtubules to modulate the recruitment of motors to the SCV (Guignot et al., 2004; Boucrot et al., 2005), to redirect vesicle trafﬁcking (Kuhle et al., 2006) or to position the SCV in the vicinity of the Golgi (Abrahams et al., 2006; Ramsden et al., 2007b). However, biochemical activities and functions for many SPI-2 effectors are undeﬁned and the molecular mechanism for how remodeling the cytoskeleton contributes to bacterial replication within the SCV is unknown. Among the SPI-2 effectors, SifA plays an essential role in bacterial replication, virulence in inbred susceptible mice, the regulation of SCV formation, and phagosome tubulation (i.e., Sifs; Stein et al., 1996; Beuzon et al., 2000). SifA translo- cated from bacteria localizes to the SCV and Sifs (Brumell et al., 2002), whereas green ﬂuorescent protein (GFP)-SifA transfected into epithelial cells show a mixed distribution in the cytoplasm, plasma membrane, and endosomal/lysoso- mal compartments (Boucrot et al., 2003; Ohlson et al., 2008). The C-terminal tail of SifA is prenylated and acylated at the CaaX sequence and is important for its function and mem- 3567