D.B.N. Vinh et al. intermediates and the vacuole (yeast lysosome; Vida and Emr, 1995). Overexpression of GFP-SifA did not appear to affect the general distribution of Abp1-CFP, Snf7-RFP, or Chc1-RFP, or the uptake and trafﬁcking of FM4-64 to the vacuolar membrane, suggesting normal endocytosis and protein sorting in these cells (data not shown). Although there was no evidence of colocalization in most cases, GFP- SifA dots transiently overlapped with Abp1-CFP (15% cells) and Snf7-RFP (10% cells) dots. However, GFP-SifA struc- tures appeared to be substantially more static compared with Abp1-RFP or Snf7-RFP structures, which were highly dynamic and had a high turnover rate as they trafﬁcked through the endocytic pathway (Kaksonen et al., 2005). The low frequency of association observed between GFP- SifA and yeast endocytic markers could be due either to the dynamic nature of certain endocytic compartments or that GFP-SifA might only trafﬁc through part of the endocytic pathway. Further analyses in mutants blocked at speciﬁc endocytic trafﬁcking steps might reveal organelle interme- diates that accumulate GFP-SifA as they trafﬁck through this step in the pathway (Bowers and Stevens, 2005). We studied the distribution of GFP-SifA in deletion mutants of genes regulating endosomal/vacuolar trafﬁcking: early endocyto- sis (abp1, sla1, sla2, ark1, prk1; Kaksonen et al., 2003); to and from late endosomes (vps21: Rab5 homologue; vps27, vps34); and to and from the vacuole (ypt7: Rab7 homologue, vps1, vps41; Bowers and Stevens, 2005; Nickerson et al., 2009). The presence of overexpressed GFP-SifA did not affect the nor- mal growth of any of these deletion mutants. As shown in Figure 1A, except for vps1 cells, the punctate distribution of GFP-SifA structures is unaffected in all trafﬁcking mutants tested. In vps1 cells, there appears to be substantially fewer and larger GFP-SifA structures, and a single tubular GFP- SifA structure was found to be anchored at the dividing neck of most large budded/G2 cells (Figure 1, A and B). To eliminate the possibility that the novel phenotype was caused by nonspeciﬁc mutations outside the vps1 deletion, a plasmid containing a single copy of wild-type VPS1 was introduced together with plasmid GFP-SifA into vps1 cells. As shown in Figure 1B, plasmid-expressed Vps1p reverts the phenotype of GFP-SifA structures from tubules to patches in vps1 cells, suggesting that GFP-SifA resides in an organelle whose distribution is speciﬁcally affected by Vps1p function. SifA Localizes to Yeast But Not Mammalian Peroxisome Membranes Vps1p is a member of the dynamin-like protein family that facilitates the scission of vesicles after budding (Praefcke and McMahon, 2004). Although the role of dynamin in yeast endocytosis remains unclear, Vps1p participates in diverse pathways including vacuolar protein trafﬁcking (Vater et al., 1992), actin organization (Yu and Cai, 2004) and peroxisome ﬁssion (Hoepfner et al., 2001). The striking GFP-SifA ﬁla- ment observed in vps1 cells is reminiscent of the elongated tubular peroxisomes previously seen in the same mutant (Hoepfner et al., 2001). To test whether GFP-SifA might be recruited to peroxisomes in wild-type cells, we cotrans- formed pGFP-SifA and pDsRed-PTS1, which contains the reporter protein RFP fused to the peroxisomal targeting sequence PTS1 at the C-terminus (Smith et al., 2002). As shown in Figure 2A, GFP-SifA punctate structures tightly colocalize with DsRed-PTS1–marked peroxisomes, suggest- ing that they reside in the same cellular compartment. Subcellular fractionation was also used to conﬁrm that GFP-SifA was stably associated with peroxisomes. GFP-SifA was expressed in a strain containing a chromosomal copy of 3570 the peroxisomal protein Pex11 fused to protein A, and cells were incubated with oleic acid to induce peroxisome prolif- eration. Whole cell lysate was centrifuged and separated into a cytosol-enriched supernatant (20KgS) and crude or- ganellar pellet (20KgP). As shown in Figure 2C, most of GFP-SifA sedimented in the 20KgP fraction. This GFP-SifA enriched material was subjected to isopycnic gradient cen- trifugation, and nearly all GFP-SifA was observed to comi- grate with fractions containing Pex11-PA (Figure 2B). We further extracted the peroxisome-enriched fractions from the gradient (no. 9 and 10, Figure 2B) to determine the subcel- lular distribution of GFP-SifA. After subjecting these frac- tions to hypotonic lysis, GFP-SifA was found predominantly in the membrane pellet (Ti8P) similar to the behavior of the membrane protein Pex11-PA (Figure 2C). The Ti8P fraction was further extracted with sodium carbonate to separate peripheral from integral membrane proteins of peroxisomes. Under this condition, virtually all GFP-SifA cofractionated with Pex11-PA in the supernatant (CO3S) enriched for pe- ripheral membrane proteins (Figure 2C). Taken together, these data indicate that GFP-SifA behaves mainly as a pe- ripheral membrane protein on yeast peroxisomes in a simi- lar manner to its behavior on mammalian cell membranes (Boucrot et al., 2003). Localization of GFP-SifA to yeast peroxisomes was a sur- prising result given its localization in mammalian cells and raised the possibility that SifA might transiently associate with peroxisomes in mammalian cells before trafﬁcking to the SCV. Therefore, SifA was analyzed for colocalization with the peroxisomal marker PMP70 in mammalian cells. On transfection of pEGFP-SifA (Boucrot et al., 2003) into epithelial CHO cells, the majority of EGFP-SifA localized to the plasma membrane and LAMP-1 positive compartment as has been reported in other cell types (Figure 3A). Inter- estingly, a small fraction of transfected CHO cells ( 10%) showed EGFP-SifA colocalizing with PMP70 (Figure 3B). Colocalization was observed as early as on day 1 and again on day 5 after transfection. However, we were unable to increase this fraction of cells through induction of peroxi- some proliferation with the drug 4-phenylbutyrate (Li et al., 2002). This pattern of colocalization was neither observed in transfection nor bacterial translocation studies of RAW mouse macrophages, HeLa human epithelial cells or pri- mary human ﬁbroblasts. We also found that Salmonella rep- lication and phagosome tubulation appeared normal in pri- mary human ﬁbroblasts from Zellweger patients that completely lack peroxisomes (data not shown). Therefore, the localization of SifA to peroxisomes was unique to yeast and SifA does not usually associate speciﬁcally with mam- malian peroxisomes. SifA Changes Yeast Peroxisome Abundance and Morphology To further understand how GFP-SifA affects peroxisome activities, we characterized the abundance and morphology of peroxisomes labeled with DsRed-PTS1 when yeast cells were incubated in medium with or without the fatty acid oleic, a condition that requires peroxisome replication in order to maintain growth. As expected, the proliferation of peroxisomes was induced 40% in cells containing a control vector grown in medium with oleic acid compared with rafﬁnose (Figures 4, A and B, and Table 1). In the absence of GFP-SifA, peroxisomes appeared punctate and distinct from one another in medium with rafﬁnose or oleic acid. The introduction of GFP-SifA has no apparent effect on the growth rate of cells in rafﬁnose and GFP-SifA colocalized tightly with peroxisomes. However, in oleic acid, GFP-SifA Molecular Biology of the Cell