D.B.N. Vinh et al. Figure 4. GFP-SifA alters the morphology of peroxisomes. Distribution of peroxisomes (DsRed-PTS1 labeled) in ﬁxed wild-type cells grown in media containing rafﬁnose (A), or oleic acid (B and C). Cells were cotransformed with pDsRed-PTS1 and empty vector (A and B) or pGFP-SifA (C). Overlay indicates merged images of ﬂuorescence and DIC; and yellow indicates the overlap of red and green signals. In C, boxed images to the left of each panel are 2.5 enlargements of smaller areas in each panel indicated by arrowheads or small white squares. Note that peroxisomes associated with SifA are less abundant, tend to clump, and are not always round in shape. The localization of SifA is to one side of peroxisomes and in many cases, appears to bridge nearby clusters of per- oxisomes (middle enlarged box). All bars, 2 m. (D) Cells containing either vector or pGFP-SifA were incubated in oleic acid for 18 h and then ﬁxed and processed for EM. Boxed images at the lower right in each panel show 2 enlarge- ment of speciﬁc areas containing peroxisomes that are depicted with letters. P, free and dis- persed peroxisomes; P*, clusters of peroxisomes with adherent membranes; P#, enlarged peroxi- somes with exaggerated membrane invagina- tions; N, nucleus; L, lipid body; M, mitochon- dria; V, vacuole. Bar, 1 m. we analyzed whether GFP-SifA affects the actin distribution around peroxisomes in pex25 or rho1 mutants. As shown in Figure 6, a higher percentage of cells in the pex25 and rho1 background compared with wild-type contains actin patches that mostly overlap with peroxisomes. These results are consistent with previous studies, suggesting that actin reor- ganization around peroxisomes depends on Rho1p and is altered in these mutants (Marelli et al., 2004). The expression of GFP-SifA in either pex25 or rho1 mutants suppressed the actin accumulating phenotype on peroxisomes (Figure 6B). GFP-SifA most likely compensates for a deﬁciency of Rho1p activity at peroxisomes in pex25 or rho1 mutants by recruit- ing Rho1p to peroxisomes in the absence of pex25 and/or potentiating its activity during rho1 depletion. DISCUSSION SifA is a type III effector protein required for Salmonella intracellular replication and virulence in mammals. In an effort to develop a genetic system to understand SifA activ- ity and function in host cells, we expressed SifA in yeast cells. SifA was recruited to yeast peroxisomal membranes with a result of fewer, large peroxisomes that contain mul- tiple membrane invaginations, suggesting hyperactive membrane constrictions without division. This phenotype may be analogous to the endosomal tubulation in mamma- lian cells caused by coexpressing SifA and another bacterial effector acting as a lipase. Furthermore, SifA appears to 3572 promote Rho1p activity on peroxisomes consistent with its ability to interact with mammalian GDP-RhoA and its struc- tural similarity to bacterial GEFs. We propose that SifA mode of action is similar in yeast and mammalian cells, but on different membrane systems perhaps by binding to a protein conserved between mammalian endosomes and yeast peroxisomes. SifA Effects on Peroxisome Division in Yeast The effects of SifA on peroxisome morphology argue for it functioning to control peroxisome division. In yeast cells, peroxisome biogenesis occurs via two pathways: de novo formation from ER precursors or growth and division of preexisting peroxisomes (Fagarasanu et al., 2007; Motley and Hettema, 2007). Division of preexisting peroxisomes in- cludes distinct steps of tubulation/elongation, constriction, and ﬁssion. Analyses of yeast peroxisome biogenesis mu- tants have separated them into gene products that affect assembly (de novo) or division (size and number). The ex- pression of GFP-SifA slightly decreases the growth on oleic acid plates of wild-type and only the division mutants (pex11 , pex25 , vps1 ), but not the assembly mutants (pex3 , pex5 , pex19 ), implicating SifA as altering the divi- sion steps of peroxisome biogenesis. By sequence similarity and overlapping functions, Pex11p, Pex25p, or Pex27p are grouped into the peroxin family (Pex11-protein family) involved in the division steps before ﬁssion, with Pex11p and Pex25p being the dominant Molecular Biology of the Cell