Deletion of JSN1 affects anterograde but not retrograde mitochondrial movement. Mitochondrial motility in yeast bearing deletions in JSN1 (KFY202), ARC18 (KFY214), ARP2 (KFY209), or temperature-sensitive mutation in ARP2 (KFY218), and the corresponding wild-type cells was evaluated using pCIT1-GFP to label mitochondria and time-lapse fluorescence microscopy. Mitochondria undergoing anterograde and retrograde movement were scored in a single focal plane, recorded as described in Materials and Methods, and graphed as a percentage of total movements (n >100). Images were acquired at 1-s intervals.

Deletion of JSN1 results in a reduction in the amount of Arp2/3 complex subunits that are recovered with mitochondria after subcellular fractionation. (A) Wild-type yeast (KFY119) and jsn1Δ cells (KFY118) that express ARC18 tagged at its chromosomal locus with GFP were grown to mid-log phase. Whole cell extracts (20 μg) and isolated mitochondria (20 μg) from these yeast cells were analyzed using Western blots and antibodies raised against GFP, Arp2p, cytochrome b₂, and Gas1p. (B) Quantitation of the amount of Arc18p-GFP, Arp2p, cytochrome b₂, and Gas1p revealed equal recovery of markers for mitochondria and plasma membrane in mitochondria isolated from wild-type and jsn1Δ cells, and a 50% decrease in the recovery of both Arc18p-GFP and Arp2p in the jsn1Δ mitochondria compared with wild-type mitochondria.

Deletion of JSN1 results in defects in mitochondrial morphology but has no effect on cytoskeletal organization or association of mitochondria with actin cables. (A) Mitochondria in midlog phase wild-type yeast (KFY206, a and c) and jsn1Δ cells (KFY202, b and d) were visualized using pCIT1-GFP and fluorescence microscopy. Images shown are two-dimensional projections of 3-D reconstructions obtained as for Figure 2. Gray outlines show cell boundaries detected by phase contrast imaging. Bar, 1 μm. (B) Microtubules were visualized in wild-type cells (KFY103, a) and jsn1Δ cells (KFY301, b) using a tubulin-GFP fusion protein. Bar, 1 μm. (C) Wild-type yeast (KFY206) and jsn1Δ cells (KFY202) expressing pCIT1-GFP were grown to mid-log phase, fixed, and stained for actin using Alexa-phalloidin. Mitochondria (a and d), actin (b and e), and an overlay showing mitochondria (green) and actin (red) (c and f) for wild-type cells (a–c) and jsn1Δ cells (d–f) are shown. Bar, 1 μm.

Jsn1 localizes to mitochondria in intact yeast. Jsn1p localizes to punctate structures that colocalize with mitochondria. Yeast (KFY206) expressing a fusion protein consisting of the mitochondrial signal sequence of citrate synthase fused to GFP (pCIT1-GFP) were grown to mid-log phase, fixed, and converted to spheroplasts. Jsn1p was visualized by indirect immunofluorescence using an affinity-purified polyclonal anti-Jsn1p antibody. Mitochondria were visualized using fluorescence imaging of pCIT1-GFP. Z-sections through the cell were collected, deconvolved, and reconstructed into a 3-D volume. The images shown are two-dimensional projections of reconstructed 3-D volumes. Mitochondria were resolved as long, tubular structures that align along the mother-bud axis (green). Jsn1p was detected in punctate cytoplasmic structures (red). The overlay of Jsn1p (red) with mitochondria (green) reveals colocalization of Jsn1p-containing particles with mitochondria (right). Gray outlines show cell boundaries detected by phase contrast imaging. Bar, 1 μm.

Jsn1p interacts with mitochondria-associated Arc18p-GFP. (A) Whole cells extracts from untagged controls (BY4743; lane 1) or yeast expressing TAP-tagged Jsn1p (2GS-B-4; lane 2) were purified using IgG-coupled Sepharose beads, as described in Materials and Methods. IgG-Sepharose beads were incubated with purified Arp2/3 complex (0.5 μg) for 2 h at 4°C. The beads were concentrated by centrifugation, and unbound Arp2/3 complex in the supernatant was removed. Beads were washed, incubated with TEV protease, and material released by protease treatment was isolated from the beads, as described above. All samples described below were analyzed using Western blots. Jsn1p-TAP_full: IgGSepharose beads after incubation with whole cell extracts from untagged or tagged cells, probed with an antibody raised against the TAP tag. Jsn1p-TAP_TEV, material released from the beads after treatment with TEV protease, probed with an antibody raised against the TAP tag. Arp2p_unbound, Arp2/3 complex that did not bind to the beads, probed with anti-Arp2p antibody. Arp2p_TEV-released, material released from the beads after treatment with TEV protease, probed with anti-Arp2p antibody. (B) Mitochondria were isolated from mid-log phase yeast cells expressing GFP-tagged Arc18p (KFY119) or untagged ARC18 (BY4743). After solubilization of mitochondria in digitonincontaining buffers, Arc18p-GFP was immunoprecipitated using protein G-Sepharose beads conjugated to polyclonal rabbit anti-GFP IgGs. Aliquots of extracted mitochondria (total) and proteins immunoprecipitated from mitochondrial extracts (pellet) were analyzed using Western blots and antibodies raised against GFP, Jsn1p, and porin.

Jsn1p colocalizes with mitochondria-associated Arc18p-GFP and is required for localization of Arc18p-GFP to mitochondria. (A) Yeast expressing ARC18 tagged at its chromosomal locus with GFP (KFY119) were grown to mid-log phase, fixed, and stained for Jsn1p as for Figure 1, and for DNA using DAPI. Z-sections through the cell were collected, deconvolved, and reconstructed into a 3-D volume. The images shown are three projections. (a) DAPI-stained material. Nuclear DNA was resolved as a single large spherical structure in the mother cell. mtDNA was resolved as punctate cytoplasmic material. b, c, and d show the localization of Arc18p-GFP (green) and Jsn1p (red) either alone (c and d) or merged (b). Arc18p-GFP was resolved as punctate structures that were enriched in the bud. Jsn1p was resolved as punctate structures that were enriched in the mother cell. Visualization of 3-D reconstructions at different rotation planes revealed that Arc18p-GFP in mother cells were associated with Jsn1p. In rotation angle shown, Jsn1p structures colocalized or overlapped with Arc18p-GFP-labeled structures. White outlines show cell boundaries detected by phase contrast imaging. Bar, 1 μm. (B) Wild-type yeast cells (KFY401, a), jsn1Δ cells (KFY404, b) expressing Arc18p-GFP and a fusion protein consisting of the mitochondrial signal sequence of subunit 9 of F₁ F0-ATPase fused to RFP were grown to mid-log phase and visualized by epifluorescence microscopy. Arc18p-GFP is shown in green and mitochondria labeled with the targeted RFP are shown in red. Z-sections through the cell were collected, deconvolved, and reconstructed into a 3-D volume. The images shown are two-dimensional projections of reconstructed 3-D volumes. Colocalization was defined as instances where two fluorescent structures either completely or partially overlapped at multiple viewing angles in rendered 3-D volumes. Deletion of JSN1 resulted in fragmentation of mitochondria and a decrease in the amount of Arc18p-GFP that colocalized with mitochondria. White outlines show cell boundaries detected by phase contrast imaging. Bar, 1 μm.

Jsn1p is a peripheral mitochondrial outer membrane protein. (A) Mitochondria were isolated from a jsn1Δ mutant (KFY200) and the corresponding wild-type cells (BY4743) by differential and Nycodenz gradient centrifugation. Recovery of Jsn1p and porin with isolated yeast mitochondria was analyzed using Western blots. (B) Wild-type cells (BY4743) were grown to mid-log phase in lactate medium and separated into subcellular fractions as described in Materials and Methods. Equal amounts of protein from S1, P1, crude mitochondrial pellet (P2), Nycodenz-purified mitochondria (NM), P3, and S3 were analyzed using Western blots and antibodies that recognize the mitochondrial marker porin, the cytosolic marker hexokinase (Hxk1p), the plasma membrane marker Gas1p, and the endoplasmic reticulum marker Sec61p. (C) Nycodenz-purified mitochondria prepared from a wild-type strain (BY4743) treated with sodium carbonate or KCl as described in Materials and Methods. Carbonate or salt-extractable and -inextractable material was analyzed using Western blots and antibodies raised against Jsn1p, porin (an integral mitochondrial membrane protein), and cytochrome b₂ (a peripheral mitochondrial inner membrane protein). T, total mitochondrial extract; P, pellet; and S, supernatant. (D) Nycodenz-purified mitochondria were washed with protease inhibitor-free breaking buffer and incubated with a protease inhibitor cocktail or trypsin and chymotrypsin (150 μg/ml) for 30 min at 23°C. After addition of protease inhibitors to the proteasetreated sample, mitochondria were separated from the reaction mixture. Proteins in the mitochondrial pellets were analyzed using Western blots and antibodies raised against Jsn1p, Tom70 (a mitochondrial outer membrane protein), and cytochrome b²(a peripheral membrane protein on the outer leaflet of the inner mitochondrial membrane).