D.B.N. Vinh et al. Figure 6. Effects of GFP-SifA on actin dynam- ics on yeast peroxisomes. (A) Distribution of actin relative to peroxisomes and GFP-SifA was analyzed by direct ﬂuorescence microscopy. Various strains containing pDsRed-PTS1 and pGFP-SifA or vector alone were grown in oleic acid for either 18 h at 30°C or 22 h at 24°C (for rho1). Cells were ﬁxed and stained with phal- loidin-Alexa660 to visualize actin. [Actin PTS1] panels show merged images with yellow areas (arrowheads), indicating colocalization of actin patches and peroxisomes in pex25 and rho1 cells. Note that in cases where GFP-SifA over- laps with peroxisomes, actin colocalization with peroxisomes is less prevalent. Bar, 2 m. (B) The frequency of cells that have overlapping actin and peroxisomes are scored for each strain. Results are from examining more than 160 cells for each strain. peroxisomes as in pex25 or rho1 cells, again suggesting that SifA largely acts by promoting Rho1p activity. SifA does not appear to improve all aspects of Rho1p function since it cannot complement the vegetative growth defects of rho1 mutants (Olson and Miller, unpublished data). SifA Role in Membrane Dynamics In yeast, peroxisome division under noninducing condition occurs mainly via replication of preexisting mature peroxi- somes, but upon induction with oleic acid, more de novo assembly might be activated to accommodate rapid prolif- eration and turnover of the peroxisome membrane system (Schrader and Fahimi, 2006; Fagarasanu et al., 2007, Smith and Aitchison, 2009). This might involve fusion of ER-de- rived preperoxisome vesicles with each other to create new peroxisomes or with preexisting peroxisomes to promote 3574 growth and division. In general, peroxisomes in rho1 and pex25 cells are smaller than those in wild type, suggesting a defect in growth and maturation. Because both Rho1p and Pex25p are observed on peroxisomes only upon oleic acid induction, they may promote division involving fusion (Smith et al., 2002, Marelli et al., 2004). It is tempting to speculate that because SifA also only induces peroxisome morphological changes when cells are grown in oleic acid, it might act more effectively in association with Rho1p and Pex25p on certain peroxisome intermediates, such as those with membrane dynamics primed for fusion. How might Rho1p and Pex25p affect peroxisomal mem- brane activity? Rho1-dependent actin dynamics are pro- posed to be important for peroxisomal budding and fusion (Marelli et al., 2004). According to the mesh hypothesis of membrane dynamics, local and transient assembly/disas- Molecular Biology of the Cell