Figure 3. Distribution of GFP-SifA transfected in mammalian CHO cells. CHO cells were transfected with pEGFP-SifA for 24 – 48 h., followed by ﬁxation, permeabilization, and immunostaining for the endosome/lysosome marker LAMP-1 and peroxisomal marker PMP70. An overlay of the corresponding images on each row is shown, with yellow color and arrowheads marking areas of overlap with GFP-SifA. Although the majority of cells show the typical codistribution of GFP-SifA and LAMP-1 in A, a small number of cells was found to have GFP-SifA distributed to PMP70 dots (B). Bar, 10 m. containing cells have fewer distinct peroxisomes than in vector containing cells (1.8 times less, p 1.6 10 13; Table 1), and many of these peroxisomes were larger and occurred in clusters (Figure 4C). On closer examination, GFP-SifA appeared to localize to one side of the peroxisome clusters in many cells, and occasionally, was found between two adja- cent clusters. Detailed EM analyses (Figure 4D and Table 2) showed that the peroxisome structures could be subdivided into two classes of morphology: 1) clusters of two or more peroxi- somes with adherent, and in some cases, thickening mem- branes (P*) and 2) enlarged peroxisomes with exaggerated and invaginated membranes (P#). Consistent with the ﬂuo- rescence data, fewer GFP-SifA containing cells were found to exhibit free and dispersed peroxisomes (category P in Figure 4D), and the average peroxisome count per cell area assayed was also substantially reduced in these cells (Table 2). In examining the EM data, although the frequency of cells showing adherent membranes (category 1 above) does not appear to be very different between vector and GFP-SifA populations, the percentage of cells exhibiting peroxisomes Vol. 21, October 15, 2010 Salmonella SifA on Yeast Peroxisomes with invaginated membrane increased strikingly from 6.3 for control, to 63 for GFP-SifA plasmid (Table 2). Therefore, at least half of the peroxisome clusters observed by ﬂuores- cence microscopy in GFP-SifA– expressing cells contained enlarged peroxisomes with invaginated membranes. SifA Activities in Yeast Mutants Defective for Peroxisome Biogenesis We sought further insight into how GFP-SifA might interact with peroxisomal proteins to affect the morphology of per- oxisomes. Mutants deleted for gene products (the peroxins) that affect peroxisome assembly (pex3 , pex19 ), size and number (pex11 , pex25 , vps1 ), or matrix protein import and maturation (pex5 , pex7 ; Fagarasanu et al., 2007; Platta and Erdmann, 2007; Smith and Aitchison, 2009), were trans- formed with pGFP-SifA and pDsRed-PTS1 and grown in oleic acid, and then cells were analyzed by ﬂuorescence microscopy. Because Pex25p acts as a peroxisomal receptor for Rho1p, we also tested GFP-SifA effects on peroxisome distribution in a temperature-sensitive rho1 mutant (Marelli et al., 2004). Overall, GFP-SifA did not improve the growth rate of any pex mutants on oleic acid plates, but caused a slight delay in growth of wild-type, pex11 , pex25 , and vps1 mutants, but not of pex3 , pex5 , pex7 , or pex19 cells. Furthermore, GFP-SifA did not appear to improve any of the peroxisomal morphological defects found in these pex mutants. Three peroxisome phenotypes were observed: 1) diffuse cytoplas- mic DsRed-PTS1 staining as in pex5 (Figure 5), pex3 , and pex19 cells. In this category, functional mature peroxisomes were not formed, causing GFP-SifA distribution to be dis- persed. Another phenotype (category 2)) included fewer, distinct peroxisomes as in pex7 (Figure 5 and Table 1). PEX5 and PEX7 encode receptors for peroxisomal matrix protein targeting sequences PTS1 and PTS2, respectively. Thus, DsRedPTS1-labeled peroxisomes would be observed in pex7 , but not in pex5 cells (Figure 5). Because GFP-SifA localization was virtually identical to DsRed-PTS1, GFP-SifA must be preferentially recruited to peroxisomes importing PTS1-speciﬁc matrix proteins. The ﬁnal peroxisomal pheno- type (category 3)) was found in vps1 (Figure 1), pex11 (Figure 5), and pex25 (Figure 6) mutants: the number of small and dispersed peroxisomes decreases and is replaced by a few large peroxisomes (Hoepfner et al., 2001; Rotten- steiner et al., 2003; Tam et al., 2003). Similarly to its behavior in a wild-type background, GFP-SifA reduced the number of free PTS1-labeled peroxisomes in pex7 and pex11 cells (statistically signiﬁcant difference between vector vs. GFP- SifA plasmid containing populations, p 0.003 and p 0.002, respectively; Table 1). Interestingly, GFP-SifA did not appear to reduce the number of free peroxisomes in a pex25 (p 0.56) or a rho1 mutant (p 0.18). Therefore, GFP-SifA activities on peroxisome morphology and abundance re- quire the presence of functional Pex25p or Rho1p. SifA Alters Actin Organization around Peroxisomes via Rho1p SifA belongs to a family of bacterial effectors that promote Ras-like GTPase activities at mammalian cell membranes (Alto et al., 2006; Ohlson et al., 2008; Huang et al., 2009). The yeast Rho1p GTPase has previously been shown to be in- volved in regulating peroxisome maturation and abun- dance. On oleic acid induction, Rho1p is recruited to peroxi- somes via Pex25p and regulates the organization of the surrounding actin cloud, thus affecting membrane dynamics during peroxisome division (Marelli et al., 2004). To further understand the role of SifA in peroxisome activities in yeast, 3571