Table 1. Fluoresence microscopic analyses of peroxisomes in wild- type and mutant cells Average no. of discrete dots/cella Vector pGFP-SifAWT 5.4 7.2 2.6 3.2 3.8 3.5 2.5 4.6 2.6 2.9 3.6 4.5 5.5 3.9 1.2 1.5 4.0 2.6 1.9 3.7b 1.8 1.6 4.2b 2.2b Strain background Without oleic acid WT With oleic acid WT pex7 pex11 pex25 rho1-4D Cells transformed with plasmids pDsRed-PTS1 and pGFP-SifAWT (pDV116) or empty vector (p426TEFpr) were grown in medium with or without oleic acid, then ﬁxed and visualized by direct ﬂuores- cence microscopy. In experiments with pGFP-SifA, PTS1-labelled peroxisomes were scored in cells expressing GFP-SifA structures. In all cases, 95% cells have overlapping DsRed-PTS1 and GFP-SifA structures. Results were scored in an average of 100 cells per strain, and each experiment was repeated at least once. WT, wild-type strain. a Average numbers of dots SD, are either dispersed or attached, but distinctly small round peroxisomes labelled with DsRed-PTS1. b Statistically signiﬁcant difference (p 0.005) was observed for the average numbers of dots between vector and pGFP-SifA popula- tions in all cases, except in pex25 (p 0.56) and rho1 (p 0.18) background. regulators (Thoms and Erdmann, 2005; Yan et al., 2005). Overproducing Pex11p promotes peroxisome proliferation and elongation, and deletion in PEX11-family genes typi- cally results in a few enlarged peroxisomes with rare occa- sion of membrane invaginations in the triple deletions (Tam et al., 2003; Rottensteiner et al., 2003; Koch et al., 2004). The peroxisome phenotype caused by SifA is unlikely to be a ﬁssion defect, which is often seen as beads-on-string peroxi- somes in the dynamin-like vps1 mutant (Hoepfner et al., 2001). What is observed by EM might be large amorphous peroxisomes with multiple arms that look like invaginations at cross sections. One interpretation is that SifA promotes membrane tubulation and/or constrictions and because ﬁs- sion activities are limited, overly large peroxisomes with multiple extensions result. Two lines of observation suggest that SifA exerts its effects on peroxisomes in conjunction with Pex25p and/or Rho1p Table 2. EM analyses of peroxisomes in wild-type cells Salmonella SifA on Yeast Peroxisomes Figure 5. Distribution of GFP-SifA in pex mutants. pex cells selected for expression of plasmids carrying GFP-SifA and DsRed- PTS1 were grown in oleic acid for 17 h and then ﬁxed for direct ﬂuorescence microscopy. PEX5 and PEX7 encode receptors for per- oxisomal targeting-signal sequences PTS1 and PTS2, respectively; PEX11 regulates peroxisome proliferation. Note that the diffuse cytoplasmic staining in pex5 indicates that no functional peroxi- somes were assembled. Overlay shows merged images of the GFP and DsRed channels, and in the bottom two cases, also overlaid with the corresponding DIC images. Bar, 2 m. activity. First, SifA reduced the number of free peroxisomes and increased the frequency of clustered peroxisomes in wild-type, pex7 , and pex11 cells, but not in pex25 or rho1 cells. Because wild-type Pex25p or Rho1p must be present for the morphological changes induced by SifA to be appar- ent, this is most likely an exaggeration of Pex25p/Rho1p activities. Other studies have reported that overproducing Pex25p can sometimes cause unusually overgrown mem- brane sheets resembling karmellae near the ER/nucleus, suggesting its role in membrane modiﬁcation (Rottensteiner et al., 2003). This would be consistent with SifA working with Pex25p/Rho1p to cause membrane invaginations. Sec- ond, SifA is able to complement the ability of pex25 and rho1 mutants to regulate actin dynamics around peroxi- somes. The fact that SifA does not cause a higher than wild-type frequency of cells with accumulating actin on peroxisomes suggests an already maximally measurable phenotype in the presence of Pex25p/Rho1p. The effects of SifA on actin is revealed only when Rho1p is deﬁcient at the Plasmid Vector pGFP-SifAWT Free and distinct (%)a 64.6 38.2 Peroxisome countb 0.30 0.07 Clusters of two peroxisomes with adherent membranesc (%) 66.7 63.2 Invaginations and exaggerated membranesc (%) 6.3 62.5 Wild-type cells selected for either pGFP-SifAWT or vector were grown in oleic acid for 18 h before ﬁxing and processing for EM. a Percent of total cells analyzed (162 for vector, and 160 for SifA); values add up to more than 100% because cells may exhibit more than one phenotype. b Number of free and distinct peroxisomes counted (437 for vector; 105 for pGFP-SifA) per m2 of total cell area assayed (1454.1 m2 for vector; 1496.7 m2 for GFP-SifA). c See Figure 4D for depiction of phenotypes. These peroxisomes are not included in the calculation of peroxisome count. Vol. 21, October 15, 2010 3573