D.B.N. Vinh et al. brane association (Boucrot et al., 2003; Reinicke et al., 2005). Structural studies indicate that SifA has two domains (Ohl- son et al., 2008). The N-terminal domain binds mammalian SKIP, which binds kinesin and links microtubules to the SCV, contributing to phagosome tubulation (Boucrot et al., 2005). SifA also competes with the late endosomal GTPase Rab9 for binding to SKIP and alters lysosomal protein traf- ﬁcking (Jackson et al., 2008). The C-terminal domain of SifA is similar to the Salmonella SPI-1 effector SopE, which func- tions as a GTPase-exchange factor (GEF) for Cdc42, Rac1, and RhoG (Ohlson et al., 2008). SifA can bind the GDP- bound form of RhoA, although no direct GEF activity has been demonstrated. Within the region of SifA structure that is analogous to the catalytic domain of SopE, is the WxxxE motif that is conserved among the family of bacterial effec- tors that targets small GTPases (Alto et al., 2006; Huang et al., 2009; Bulgin et al., 2010). Interestingly, Sif-like structures are generated when the SPI-2 effector SseJ is ectopically coex- pressed in HeLa cells with either SifA or activated RhoA, B, or C, suggesting that SifA might function as a GEF to acti- vate Rho proteins (Ohlson et al., 2008). SseJ is a lipase that is activated by RhoA in vitro and can esterify cholesterol in mammalian cell membranes, possibly contributing to mem- brane tubulation (Christen et al., 2009). As a complementary approach to the challenging task of correlating biochemical activities of individual SPI-2 effec- tors with the cellular phenotypes seen in mammalian cells, we characterized SifA activity in the model organism Sac- charomyces cerevisiae. Budding yeast has proven very useful for studying the interactions of bacterial effectors with host targets and gaining insight into their enzymatic functions (Valdivia, 2004; Siggers and Lesser, 2008; Curak et al., 2009). The pathways of protein trafﬁcking, cytoskeletal organiza- tion, and signal transduction are well studied in yeast, and bacteria recognize analogous components in many steps of these conserved cellular processes. For example, bacterial effectors that alter the actin cytoskeleton have been shown to target yeast Rho1p or other components of the Rho family pathways when they are expressed in yeast (Von Pawel- Rammingen et al., 2000; Alto et al., 2006). We report here that SifA is recruited to the membrane domain of yeast peroxi- somes and most likely promotes peroxisome-speciﬁc Rho1p activities which include regulating actin organization. Thus, SifA may be used as a tool to study the biology of yeast peroxisomes, and in return, the yeast peroxisome provides a model for studying the biochemical and functional proper- ties of SifA interactions with Rho GTPases at the membrane. MATERIALS AND METHODS Yeast Strains and Plasmids All S. cerevisiae strains used in this study were derivatives of either BY4743 (MATa/ ), BY4742 (MAT ) or BY4741 (MATa) and the corresponding deletion strain library (Winzeler et al., 1999), unless otherwise indicated. Strain DVY119 (MATa ABP1-CFP::KanMX6 GAL10prGFP-SifA::LEU2 can1 his31 leu2 ura3 lys2) was created by integrating CFP (cyan ﬂuorescent protein) at the C-terminus of ABP1 in MOY14 (gift of M. Ohl, Miller laboratory) according to Wach et al., (1997). Strain MOY14 contains an integrated copy of GAL10prGFP- SifA in the W303 background and was generated as described in Lesser and Miller (2001). Strains containing Snf7-monomeric red ﬂuorescent protein (mRFP) and Chc1-mRFP are gifts of E. O’Shea (Harvard). The PEX11-pA haploid strain (YOL14MC) contains a genomically encoded Pex11p tagged at the C-terminus with Staphylococcus aureus protein-A and was created in the BY4743 background (Smith et al., 2002). Strain rho1-4D contains the Ts allele rho1-104 that was backcrossed into the BY4742 background (Marelli et al., 2004). The following plasmids were used in this study: pFusGAL10prGFP-SifA (pMO32, gift of M. Ohl) with GFP-SifA expression induced by galactose, contains SifA open reading frame (ORF) that was PCR-ampliﬁed from S. typhimurium chromosomal DNA and cloned in frame with the C-terminus of GFP in pFUSGAL10prGFP (Lesser and Miller, 2001); p426TEFprGFP-SifA 3568 (pDV116) with GFP-SifA constitutively expressed, generated by PCR ampli- fying the GFP-SifA fragment from pMO32 and introducing it into p426TEFpr (Mumberg et al., 1995) via homologous recombination; pVPS1 (pCKR19; (Vater et al., 1992); pDsRed-PTS1 contains the Discosoma sp. RFP gene FP583 fused to the peroximal signal sequence PTS1 (Ser-Lys-Leu) at the C-terminus and ﬂanked at both ends with FAA2 regulatory sequences (Smith et al., 2002). Yeast Media Synthetic minimal media (SM) contained the following: 0.17% yeast nitrogen base (YNB) without amino acids, 0.5% NH4SO4; Saccharomyces Cerevisiae induction medium (SCIM), 0.7% YNB plus NH4SO4, 0.5% yeast extract, 0.5% peptone, and 1/10 amount of amino acids. Microscopy Strains containing plasmids were routinely maintained in SM supplemented with the proper amino acids and carbon source. For expressing pFUS-based genes in trafﬁcking mutants, an overnight culture grown in the same nonin- ducing media was ﬁrst diluted into fresh SM plus 2% rafﬁnose and grown at 25°C until early log at OD600 0.2. Then, cultures were induced by adding 2% galactose and grown for another 2–3 h until midlog OD600 0.5– 0.6. Because the distribution of GFP-SifA on peroxisomes was found to be more robust in media containing rafﬁnose than in glucose, cells were routinely cultured overnight in media containing 2% rafﬁnose before oleic acid induction. We routinely use single colonies from fresh transformation of pDV116 to avoid possible revertants resulting from constitutive expression of GFP-SifA. All oleic acid induction studies were performed at 30°C, unless otherwise indi- cated. To induce peroxisomes, an overnight culture was ﬁrst diluted into fresh SM plus rafﬁnose and grown until midlog (2 107 cells/ml). Cells were then pelleted and resuspended into fresh SM or SCIM containing 0.1% rafﬁnose, 0.15% oleic acid, and 0.2% Tween-40 and incubated at 30°C for 16 –18 h (ﬁnal OD600 0.5– 0.7). For optimal viability, rho1TS cells were induced at 24°C for 20 –22 h in SCIM plus 0.1% rafﬁnose, 0.15% oleic acid, and 0.2% Tween-40. At the appropriate time points, cells were collected and visualized either live by direct ﬂuorescence microscopy, or ﬁxed ﬁrst in 2% paraformaldehyde (Ted Pella, Irvine, CA) at room temperature (RT) for 10 min, followed by several washes in a 1.2 M sorbitol and 0.1 M potassium phosphate buffer (pH 7.5). To visualize actin, ﬁxed cells were washed in PBS plus 1 mg/ml BSA, and stained with 3.3 M of AlexaFluor 660-phalloidin (Invitrogen) for 30 min at RT. For either live or ﬁxed-cell imaging, a small cell suspension was dropped onto a glass slide, and a coverslip was gently applied on top. All image acquisition were kept to under 30 min to prevent sample dehydration. Imaging was performed at RT on a Nikon Eclipse TE2000E inverted microscope (Melville, NY) with a Planapochromat 100 /1.4 oil objective. Microscope functions were controlled by the Metamorph software (Molecular Device, Sunnyvale, CA). For each sample, ﬁve z-axis planes spaced by 0.8 m were acquired. For each plane, separate images at the GFP, rhodamine, or Cy5 channel were taken with the appropriate exposure times. The planes from each channel were then merged into a single plane containing the maximal signal intensity (Stack Arith Max, Metamorph) and processed using a two-dimensional decon- volution algorithm (Nearest Neighbor 2D Decon, Metamorph). EM analyses were performed on cells induced for 18 h in SCIM plus 0.1% rafﬁnose, 0.15% oleic acid, and 0.2% Tween-40 at 30°C. Sample preparations, observations, and analyses are as previously described in Vizeacoumar et al. (2003). Subcellular Fractionation and Extraction of Peroxisomes Peroxisome isolation was based on Marelli et al. (2004) with modiﬁcations. Brieﬂy, overnight culture of yeast cells harboring PEX11-PA and p426TEFprGFP-SifA (pDV116) were induced in SCIM 0.1% rafﬁnose, 0.15% oleic acid, and 0.2% Tween-40 for 17 h at 30°C. Cells were harvested, sphero- plasts were generated, and homogenized to produce a whole cell lysate in MES buffer (5 mM MES, pH 5.5; 1 mM EDTA, 1 mM KCl) plus 0.6 M sorbitol. The whole cell lysate was subjected to a 20,000 g spin to yield a supernatant (20KgS) and a pellet (20KgP). The 20KgP material containing most of GFP- SifA, was resuspended in MES/sorbitol buffer and 0.3 mg from this sus- pension was applied to an 11-ml step gradient consisting of 17, 25, 35, and 50% Nycodenz (Axis-Shield, Norton, MA) in MES buffer. Organelles were separated by isopycnic centrifugation at 107,000 g for 4.5 h at 4°C in a SW41 rotor. Fractions of 1 ml were collected from the top of the gradient, TCA- precipitated, and analyzed by SDS-PAGE and Western blotting. Anti-GFP was a gift from A. Merz (U. of Washington), and anti-PA (PAP) was pur- chased from Sigma (St. Louis, MO). To extract peroxisomes, a portion of fraction 9, 10, or 11 enriched for peroxisomes from the Nycodenz gradient (Figure 2B) was ﬁrst diluted in MES/sorbitol buffer, and the organelles were pelleted at 20,000 g for 1 h in an SW40 rotor. The supernatant was removed, and the pellet was incubated on ice for 1 h in Ti8 buffer (10 mM Tris, pH 8; 1 mM EDTA) containing protease inhibitors. After a 200,000 g spin for 1 h in a TLA100 rotor (Beckman, Fullerton, CA), the supernatant (Ti8S) was separated from the pellet (Ti8P). The Ti8P pellet was further extracted by suspending in 0.1 M Na2CO3, pH 11.2, and incubated for 1 h on ice, followed by centrifugation at 200,000 g for 1 h to yield a supernatant (CO3S) and pellet (CO3P). Samples Molecular Biology of the Cell