The WD-repeats of Net2p interact with Dnm1p and Fis1p to regulate division of mitochondria

Abstract

The Net2, Fis1, and Dnm1 proteins are required for the division of mitochondria in the yeast Saccharomyces cerevisiae. Net2p has an amino-terminal region that contains predicted coiled-coil motifs and a carboxyl-terminal domain composed of WD-40 repeats. We found that the amino-terminal part of Net2p interacts with Fis1p, whereas the carboxyl-terminal region interacts with both Dnm1p and Fis1p. Overproduction of either domain of Net2p in yeast cells poisons mitochondrial fission, and the dominant-negative effect caused by the WD-repeats of Net2p is suppressed by increased levels of Dnm1p. Point mutations in the WD-region of Net2p or in the GTPase region of Dnm1p disrupt the normal Net2p-Dnm1p interaction, causing Net2p to lose its normal punctate distribution. Our results suggest that Dnm1p interacts with the WD-repeats of Net2p and in a GTP-dependent manner recruits Net2p to sites of mitochondrial division. Furthermore, our results indicate that Net2p is required for proper assembly of the mitochondrial fission components to regulate organelle division.

Figure 1.

Net2p contains two functional domains. (A) The Net2 protein contains predicted coiled-coil regions (amino acids 129–150 and 230–300, hatched boxes) and WD-40 repeats (amino acids 386–714, speckled box). The Net2Np and Net2WDp domains that were used in this study are denoted. (B) Dnm1p interacts with the carboxyl-terminal WD-40 repeats of Net2p in the yeast two-hybrid assay. Relative β-galactosidase activities of a GAL1::lacZ reporter in yeast cells carrying pOAD-Dnm1p in combination with pOBD-Net2p, pOBD-WDp, or pOBD-Net2Np are shown. As controls for self-activation, cells expressing only pOBD-Net2p, pOBD-WDp, or pOBD-Np were also tested. The averages of three independent experiments are shown with error bars of one SD. (C) Fis1p interacts with both the amino-terminal and carboxyl-terminal domains of Net2p. β-galactosidase activities of cells with pOAD-Fis1p and pOBD-Net2p, pOBD-WDp, or pOBD-Net2Np are shown as in B. (D) Dnm1p and the WD-repeats of Net2p interact in vivo. WT cells (FY833) carrying pHS15 (pGAL1-DNM1-HA) and either pKC65 (pGAL1-GFP-NET2N) or pKC68 (pGAL1-GFP-WD) were lysed, and total proteins were immune-precipitated with HA antibodies. One hundred percent of the supernatant (S) and pellet (P) were analyzed by SDS-PAGE followed by Western blotting with Dnm1p or G3DPH for controls and GFP antisera to detect GFP-Net2Np or GFP-Net2WDp. Sixty percent of total protein input (I) from Net2Np-containing extracts and 32% of input from Net2WDp-containing extracts were used.

Figure 2.

Overproduction of Net2p, Net2Np, or Net2WDp inhibits mitochondrial division. (A) Overproduction of Net2p. Wild-type strain FY833 containing pKC62, a vector that carries galactose-inducible GFP-Net2p, were pregrown on raffinose-containing medium and then shifted to galactose for the indicated times. Cells were viewed using fluorescence microscopy after staining with mitofluor red 589, and representative cells showing merged images from the red (mitochondria) and green (GFP-Net2p) channels are shown. Beneath each frame is the level of Net2p calculated from Western blotting with Net2p antibody. Bar, 3 μm. (B) Overproduction of Net2Np or Net2WDp. WT cells containing either pKC64 (pGAL1-GFP-Net2Np) or pKC67 (pGAL1-GFP-Net2WDp) were pregrown in raffinose medium (t = 0) and then shifted to galactose (t = 180′). Cells also carry plasmid, pHS78, which produces the matrix-targeted cox4-dsRed.T1 protein. Images of cells (DIC) and the dsRed-containing mitochondria (mito) are shown. Beneath each frame is the level of Net2p determined by Western blotting. Bar, 3 μm. (C) Quantitation of mitochondrial morphology of wild-type cells overexpressing GFP-Net2p, GFP-Net2Np, or GFP-Net2WDp. WT cells containing pKC62, pKC64, or pKC67, or the empty vector, pRS314GU, were observed after 180 min of growth in galactose medium. The mitochondrial morphology of more than 100 cells of each strain was determined.

Figure 3.

Localization of Net2Np and Net2WDp. (A) Net2Np is mitochondrial; Net2WDp is cytosolic. WT cells of strain FY833 containing pKC64 (pGAL1-GFP-Net2Np) or pKC67 (pGAL1-GFP-Net2WDp) were grown in galactose medium for 120 min. Cells were homogenized (H), and separated by centrifugation into a low-speed pellet (P₁), a mitochondrial fraction (M), and crude cytosol (C). After SDS-PAGE, fractions were analyzed by Western blotting using antibodies to GFP (GFP-Net2Np or GFP-Net2WDp), hexokinase (Hex), and Tim23p. (B) Net2Np colocalizes with mitochondria; Net2WDp is diffusely distributed throughout the cytosol. net2Δ cells expressing GFP-Net2p from plasmid pKC62, or WT cells expressing GFP-Net2Np or GFP-Net2WDp from plasmids pKC64 or pKC67 were examined by fluorescence microscopy. Mitochondria were visualized by staining with mitofluor red 589. Bar, 3 μm. (C) Net2Np requires Fis1p for its mitochondrial localization. Cells were fractionated as in part a and homogenate (H), high-speed supernatant, crude cytosol (C), and mitochondria (M) were analyzed. (D) Location of GFP-Net2Np expressed from pKC64 for 90 min was examined in fis1Δ cells using fluorescence microscopy. Bar, 3 μm. (E) Net2p requires Fis1p for its stability. Whole cell extracts from WT, dnm1Δ (dΔ), fis1Δ (fΔ), and net2Δ (nΔ) were analyzed by SDS-PAGE and Western blotting with antibodies to Dnm1p (top) and Net2p (arrow, bottom).

Figure 4.

Overproduction of Dnm1p suppresses the dominant-negative effect of Net2WDp. (A) Cells expressing Net2WDp in combination with high levels of Dnm1p contain nearly wild-type mitochondria. Strain FY833 containing pKC68 (pGAL1-GFP-Net2WDp) was transformed with pRS314GU (empty vector) or pHS15 (pGAL1-Dnm1p-HA). Cells were viewed by DIC and fluorescence microscopy after growth on galactose medium for 180 min. Mitochondria were visualized by staining with mitofluor red 589. Beneath each frame, the level of Dnm1p overexpression is shown. Bar, 5 μm. (B) Localization and distribution of Net2WDp changes when Dnm1p is overexpressed. Wild-type (FY833) cells expressing GFP-Net2WDp from pKC67 were examined by fluorescence microscopy (Net2WDp + empty vector). Wild-type cells expressing both Dnm1p and GFP-Net2WDp from the GAL1 promoter were stained with mitofluor red 589 and examined using the Delta Vision system (Net2WDp + Dnm1p). The image shown is deconvolved, projected data that was rotated 180° around the y-axis. Bar, 5 μm.

Figure 6.

Net2WDp, but not Net2WDp^R461A, coimmune precipitates with Dnm1p-HA. dnm1Δ cells containing pHS15 (pGAL1-DNM1-HA) with either pKC68 (pGAL1-GFP-NET2WD) or pKC85 (pGAL1-GFP-NET2WD^R461A) were lysed, and proteins were precipitated with HA antiserum. As a control, reactions were also incubated with protein A-sepharose alone. One hundred percent of supernatant (S) and pellet (P) fractions were analyzed by Western blotting, using GFP antibodies to detect GFP-Net2Np and GFP-Net2WDp. 30% of the total protein input (I) was used.

Figure 5.

Mutations in the WD-repeats of Net2p disrupt the Net2p-Dnm1p interaction. (A) Alignment of the WD-repeats of Net2p with Gβ1 and Tup1. Each WD repeat of the three proteins is listed on a separate line (1–7) and each β-strand of the propeller (a, b, c, d) is indicated below the protein sequences. The WD-repeats begin with the d strand of the 7th repeat, which is found immediately before the 1st WD repeat in the primary sequence. Amino acids of Net2p that were tested for interaction with Dnm1p are blue. Amino acids of Gβ1 that bind either Gγ and/or Gα are green, as are the residues of Tup1 that interact with Matα2. (B) Arginine 461 of Net2p is required for Dnm1p-Net2p interaction by two-hybrid β-galactosidase activities of cells with pOAD-Dnm1p and either pOBD-WD^F400A, pOBD-WD^R461A, pOBD-WD^D664A, or pOBD-WD^S689A are shown. The average of three independent experiments with one SD is shown. (C) Mutations in the WD-repeats impairs Net2p function and redistributes GFP-Net2p. net2Δ cells were transformed with pKC80, pKC81, pKC82, or pKC83, which express galactose-inducible fusions between GFP and Net2p^F400A, Net2p^R461A, Net2p^D664A, or Net2p^S689A, respectively. Localization of Net2p mutants was observed using DIC and fluorescence microscopy. Mitochondria were visualized by staining with mitofluor red 589. Bar, 3 μm.

Figure 7.

Mutations in Dnm1p's GTPase region interfere with Net2p-Dnm1p interaction. (A) Dnm1p GTPase mutants do not interact with Net2p by two-hybrid β-galactosidase activities of cells with pOBD-WDp, and pOAD-Dnm1p, pOAD-T62F, or pOAD-S42N are shown. The averages of three separate experiments with error bars of one SD are shown. (B) Dnm1p GTPase activity is required to form Net2p puncta. dnm1Δ cells expressing GFP-Net2p from pKC62 in combination with either Dnm1^S42N or Dnm1^T62F from pKC78 or pKC79 were examined by fluorescence microscopy. Bar, 3 μm. (C) Dnm1p localization and distribution require an intact GTPase domain. WT cells expressing fusions between GFP and either Dnm1^T62F or Dnm1^S42N from plasmids pKC76 or pKC75 were examined using fluorescence microscopy. Bar, 3 μm.

Figure 8.

Deletion of FIS1 or overproduction of Net2WDp causes Dnm1p to accumulate on mitochondria. (A) Lack of Fis1p changes the distribution of Dnm1p. dnm1Δ, net2Δ, fis1Δ, and net2Δ fis1Δ cells expressing Dnm1p-GFP from pHS20 were examined by fluorescence microscopy after staining mitochondria with mitofluor red 589. Bar, 3 μm. (B) Expression of Net2WDp causes Dnm1p to aggregate on mitochondria. Wild-type strain FY833 expressing Dnm1p-GFP from pHS20 and carrying either pKC68 (pGAL1-Net2WDp) or the empty vector pRS314GU were grown in galactose medium for 180 min. Cells were viewed by fluorescence microscopy. Bar, 3 μm. (C) A significant amount of Dnm1p cofractionates with mitochondria in either fis1Δ or Net2WDp-expressing cells. WT cells containing pRS316GU (empty vector) or pKC71 (pGAL1-Net2WDp) as well as fis1Δ cells with no plasmid were grown in galactose for 120 min. Cells were homogenized (H) and separated into a high-speed supernatant (C) and a mitochondrial pellet (M) by centrifugation. After SDS-PAGE, fractions were analyzed by Western blotting with antibodies to Dnm1p, Tim23p, and hexokinase (Hxk).