Figure 1. Distribution of GFP-SifA in endocytic trafﬁcking yeast mutants. (A) The distribution of GFP-SifA in ﬁxed wild-type or endocytic trafﬁcking deletion mutants was observed by direct ﬂuo- rescence microscopy. Cells containing plasmids that expressed ei- ther the GFP-SifA fusion (pDV116) or GFP control were grown in media containing rafﬁnose at 25°C. In all cases, except for vps1 cells, GFP-SifA localizes to distinct punctate structures (indicated by arrows). The diffuse cytoplasmic and nuclear ﬂuorescence back- ground was also observed in cells expressing only GFP. In vps1 mutants that lack the dynamin gene, GFP-SifA localizes to thin ﬁlamentous structures (arrowheads) anchored at the neck of large budded (G2) cells. Bar, 4 m. (B) Introducing a plasmid expressing the wild-type dynamin VPS1 into vps1 cells was sufﬁcient to redistribute GFP-SifA to punctate structures. Note that vps1 cells at other budding stages that harbor the control plasmid (vec) contain fewer (0 –1 dot) and larger GFP-SifA punctate structures than those with the pVPS1 plasmid. Expression of GFP-SifA (pMO32) in vps1 cells was induced in galactose for 3 h, and ﬂuorescent images were captured live. Bar, 4 m. were TCA-precipitated, and equivalent portions were subjected to SDS-PAGE and Western blot. Mammalian Cells Transfection and Immunoﬂuorescence Reagents. Plasmid enhanced green ﬂuorescent protein (pEGFP)-SifA (gift from S. Meresse Université de la Méditerranée, Marseille, France) contains the SifA ORF fused to the C-terminus of EGFP (Boucrot et al., 2003). UH1 LAMP1 mAb (1:20 dilution) was from the Developmental Studies Hybrid- oma Bank (University of Iowa, Iowa City); rabbit anti-PMP70 (1:1000) was from Invitrogen. Immunoﬂuorescence. Chinese hamster ovary (CHO)-K1 ﬁbroblasts (ATCC, Manassas, VA) were grown in F12 medium (Invitrogen) supplemented with 10% FBS, penicillin/streptomycin, at 37°C in 5% CO2. Cells were transfected with Fugene6 (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s instructions in eight-well chamber slides (Nalge Nunc International, Rochester, NY). Cells were ﬁxed in 3% paraformaldehyde for 15 min at 37°C and then permeabilized and blocked in 0.2% saponin and 5% normal goat serum in PBS for 30 min at RT. Primary antibodies diluted in blocking solution were added to cells and incubated for 2 h at RT or overnight 4°C. After three PBS washes, cells were incubated with appropriate Alexa Fluor– conjugated secondary antibodies (Invitrogen) for 30 min. Cells were again washed and mounted on coverslips with Fluoromount (Southern Bio- technology Associates, Birmingham, AL). Cells were observed under light ﬂuorescence microscope with a Planapochromat 60 /1.4 oil objective lens. Vol. 21, October 15, 2010 Salmonella SifA on Yeast Peroxisomes Figure 2. SifA distributes to the peripheral membrane of peroxi- somes in yeast. (A) GFP-SifA (pDV116) colocalizes with the perox- isomal reporter DsRed-PTS1 in wild-type yeast cells as indicated by yellow in the merged image. Cells containing two plasmids express- ing the indicated fusion reporters were grown in media containing rafﬁnose and then ﬁxed and analyzed by direct ﬂuorescence micros- copy. Bar, 4 m. (B) GFP-SifA cofractionates with the peroxisomal marker Pex11-PA by isopycnic centrifugation. Cells containing a chromosomal copy of Pex11-PA were transformed with pDV116 and induced in oleic acid for 17 h before biochemical fractionation. GFP-SifA– enriched material derived from the 20KgP crude organel- lar fraction (in C) was applied to a Nycodenz gradient and sepa- rated by centrifugation. Gradient fractions were collected from the top (fraction 1), and equal portions of each fraction were analyzed by Western blotting using anti-GFP and anti-pA antibodies. (C) Materials from fraction 10 were further extracted in a low-salt buffer to separate matrix (Ti8S) from membrane (Ti8P) peroxisomal pro- teins. The Ti8P fraction was again extracted with Na2CO3 to distin- guish between peripheral membrane (CO3S) and integral mem- brane (CO3P) proteins. Equivalent portions of each fraction were analyzed by Western blot. Note that GFP-SifA and Pex11-PA be- have mainly as peripheral membrane proteins. RESULTS SifA Distribution Is Not Associated with Yeast Endosomal Compartments The intracellular distribution of SifA upon expression in yeast was ﬁrst analyzed to determine whether it would be localized to endosomal compartments similar to its localiza- tion in mammalian cells (Brumell et al., 2002; Boucrot et al., 2003). A fusion protein construct containing SifA linked to GFP at the N-terminus was created similarly to the GFP-SifA construct previously characterized in mammalian cells (Bou- crot et al., 2003). The expression of GFP-SifA was tested under either constitutive expression or under the control of an inducible promoter, but similar growth rates were ob- served in all media for either mode of expression. GFP-SifA localized to distinct punctate structures throughout the cell in a wild-type strain, in addition to the diffuse cytoplasmic and nuclear background distribution that is also observed in cells expressing only GFP (Figure 1A). The punctate staining of GFP-SifA is reminiscent of that found for yeast endosomal structures or the Golgi. Therefore, we sought to determine the identity of the GFP-SifA structures by looking for their codistribution relative to other yeast endocytic and Golgi markers: Abp1-CFP (early endocytic vesicle; Kaksonen et al., 2003), Snf7-RFP (late endosome; Huh et al., 2003), Chc1-RFP (late endosome/Golgi; Kaksonen et al., 2005; Newpher et al., 2005), and the vital dye FM4-64, which stains endocytic 3569