Mmr1 and Vac17 binding sites overlap on Myo2. (A) Mutations on the Myo2 CBD surface reveal overlapping binding regions essential for interaction with Vac17 and Mmr1. Top left squares show empty vector controls. (B) Surface (left) and ribbon (right) view of Myo2 CBD. Blue: Vac17 only; red: Mmr1 only; purple: Vac17 and Mmr1; dark gray: Sec15 binding site. (C) Mutations did not affect the levels of Myo2. (D) Myo2 mutants affecting Mmr1 and/or Vac17 interactions grow on glucose (YPD [yeast extract, peptone, dextrose]) and glycerol (YPG [yeast extract, peptone, glucose]). (E) myo2-D1297N is defective in vacuole inheritance, myo2-I1308A and -K1312A are defective in mitochondria inheritance, and myo2-L1301R and -N1304D are defective in mitochondria and vacuole inheritance. For mitochondria, strains with pmitoGFP (green) were grown in SC minus leucine media for at least six doubling times; density of <5 × 10⁶ cells/ml. For vacuoles, cells were pulsed with FM4-64 for 1 h and chased for one doubling. (F) Mitochondria inheritance was scored in small and medium budded cells according to position of mitochondria in the bud. Categories I and II were mutant and grouped together (dark bars). Categories III and IV were wild type–like and grouped together (green bars). n = 2; ≥200 cells per strain. (G) Cells scored for the presence or absence of vacuoles in small and medium budded cells. n = 3; ≥200 cells per strain. Error bars represent SDs. (H) Vacuoles (FM6-46) and mitochondria (mitoGFP) in wild-type, vac17Δ, and mmr1Δ yeast. AD, activation domain; BD, binding domain. Bars, 5 µm.

Mmr1 and Vac17 concentrate at the leading edge of inherited mitochondria and vacuoles, respectively. (A–E) Vac17-3×GFP (A and B) and Mmr1-GFP (C–E) expressed from their endogenous loci. (A) Vac17 (green) concentrated at the leading edge of the vacuole (red). Small (S), medium (M), and large (L) budded cells. Small budded (top graph; n = 34) and large budded (bottom graph; n = 31) cells were scored. When GFP was detected, it was exclusively on the vacuole membrane. Arrowhead indicates an example of Vac17 accumulation at the mother–bud neck. (B) Vac17-3×GFP is elevated and on the mother vacuole membrane in myo2-D1297N and myo2-N1304D mutants. myo2Δ cells with the indicated myo2 mutant were expressed from a plasmid (CEN and HIS3) as the sole copy of MYO2. (A and B) Before FM4-64 labeling, cells were grown in rich media for five doubling times at a density of <5 × 10⁶ cells/ml. Cells were chased for one doubling. (C) Mmr1 (green) is concentrated on the leading edge of mitochondria (red; mitoRFP expressed from a plasmid). Small budded (top graph; n = 32) and large budded (bottom graph; n = 30) cells were scored for polarized GFP signal or a diffuse GFP signal on the mitochondria membrane. Arrowheads indicate examples of Mmr1 signal at the mother–bud neck region. Arrows show mitochondria at the rear of the mother cell. (A and C) Three independent experiments. (D) Mmr1-GFP is elevated and localized throughout the mitochondria membrane in myo2-N1304D and myo2-I1308A mutants. myo2Δ cells with the indicated myo2 mutant or wild type expressed from a plasmid (CEN and HIS3) as the sole copy of MYO2 were transformed with pmitoRFP. (E) Western analysis of Myo2 in Mmr1-GFP cells containing MYO2 or myo2 mutant alleles. Anti-Pgk1 is the loading control. Bars, 5 µm.

Vacuole and mitochondria inheritance occurs early in the cell cycle at similar but not identical times. (A) Vacuole and mitochondria inheritance occurs at similar times. A pair of bars represents one cell. Bar length indicates total time of the time lapse. t = 0, time the first organelle (top bar) entered the bud. Closed arrowheads show time the second organelle entered the bud. Right columns indicate time between images. V or M indicates the number of times a vacuole (V) or mitochondria (M) crossed the mother–bud neck for the two time-lapse series with <60 s between images. Wild-type cells were transformed with pmitoGFP, grown in log phase for a minimum of six doublings, labeled with FM4-64, and chased for one doubling. Small buds initially devoid of mitochondria and vacuoles, with both mitochondria and vacuoles visible in the region around the mother–bud neck, were analyzed. Images for all acquired time-lapse series are presented in the JCB DataViewer. (B) Images of A (I; top red–green pair) as mitochondria and vacuoles crossed the mother–bud neck. Images were taken every 20 s for 13 min. Closed arrowheads show organelle in the bud. Exp s, seconds of exposure. Bar, 5 µm.

Myo2 mutations that disrupt only Vac17 or only Mmr1 binding exhibit a delay in vacuole or mitochondria inheritance, respectively. Vacuoles (FM4-64) and mitochondria (mitoGFP) in cells with myo2 mutants expressed from plasmids (CEN and HIS3) as the sole copy of MYO2. (A) t = 0, time that the first organelle (top bar) entered the bud. Arrowheads show the time that the lagging organelle entered the bud. (top) Five of six cells containing myo2-I1308A have a delay (I–III) or absence (IV and V) of mitochondria inheritance. VI was wild type–like. (bottom) Cells containing myo2-D1297N had no vacuole inheritance (VII–XI). (right) Time images per series. V or M indicates the number of times a vacuole tubule (V) or mitochondria tubule (M) crossed the mother–bud neck in series with <60 s between images. For myo2-I1308A, black numbers indicate the vacuole is inherited, whereas gray indicates a defect in mitochondria inheritance. For myo2-D1297N, black numbers indicate that mitochondria are inherited, whereas gray indicates a defect in vacuole inheritance. Dashed lines show no mitochondrial or vacuole tubule detected in the bud during the time course. Images for all acquired time-lapse series are presented in the JCB DataViewer. (B) Time-lapse images of A (IV). Arrow shows a mitochondrial tubule remaining in the mother cell, near the mother–bud neck. (C) Time-lapse images of A (VIII). (B and C) Closed arrowheads show vacuole or mitochondria tubule in bud. Exp s, seconds of exposure. Bars, 5 µm.

Vac17 competes with Mmr1 for binding Myo2 in vivo. (A) Mitochondria and vacuole inheritance measured with FM4-64 and mitoGFP, respectively. Small budded cells (bud diameter ≤1/3 mother diameter) scored for relative levels of fluorescence in the bud versus the mother. (right) Example of linear intensity scales for mitochondria and vacuoles in a wild-type small budded cell. For each cell, background was subtracted, and the bud–mother ratio of fluorescence at 528 nm (GFP) and 617 nm (FM4-64) was determined. Criteria for cells analyzed were as follows: (a) Bud in focus. (b) Both vacuoles and mitochondria visible in the mother cell. (c) Cell free of surrounding cells on at least two sides. Error bars represent SEMs; Student’s t test analysis. *, P < 0.05; ***, P < 0.001. n = 3; a minimum of 33 small budded cells per strain, per experiment; 100 total cells per strain. Dotted box of the acquired image corresponds to the same area as shown in the GFP signal box and FM4-64 signal box. Bar, 1 µm. (B) Quantitative fluorescence analysis of large budded/unbudded cells; 33 cells per strain, per experiment. Error bars show SEMs of three independent experiments, with the exception of myo2-I1308A (mitoGFP) in which two experiments are indicated (diamonds). (C) Western blot of cells transformed with empty vector, pMMR1-V5, or pVAC17 expressed from multicopy plasmids grown in log phase. (D–F) Overexpression of Vac17 or Mmr1 leads to reduced inheritance of mitochondria or vacuoles. (D) Mitochondria and vacuole inheritance assessed via mitoGFP and FM4-64. Error bars indicate SDs. (E and F) Fluorescence microscopy of wild-type cells transformed with pmitoGFP (LEU2) plus either pRS416 (empty vector), pMMR1-V5 (ADH1 promoter and URA3), or pVAC17 (ADH1 promoter and URA3). Cells were grown at least six doubling times before FM4-64 labeling. Arrows show mitochondria accumulation in the bud. Arrowhead shows accumulation of mitochondria in the mother cell at the mother–bud neck. Asterisk shows accumulation of vacuoles in the mother cell at the mother–bud neck. Bars, 5 µm.

Vac17 competes with Mmr1 for access to Myo2 in vitro. (A and B) Size-exclusion chromatography (Superdex 200, analytical column). Myo2 CBD and MBP-Vac17(112–157) (1:1 molar ratio; red line); MBP-Mmr1(387–430) (1:1 molar ratio; green line). (inset) Fractions on 10% SDS-PAGE gels. Purple arrowheads show the peak. Black and purple arrowheads show fractions tested on SDS-PAGE. Molecular mass is indicated. Elutions of Myo2 CBD monomer (gray dashed line) and MBP (42 kD) with Myo2 CBD (50.5 kD; gray dotted line). (C) MBP-Mmr1 competes with MBP-Vac17 for binding Myo2 CBD. Equal molar ratios of MBP-Mmr1 peptide (11.7 nmol) and Myo2 CBD (11.4 nmol) were added with increasing amounts of MBP-Vac17; 4.8, 9.3, 15, and 19 nmol (colored wedge) in separate runs. (top) Elutions. Gray dotted line shows MBP-Vac17 plus MBP-Mmr1 (1:1 molar ratio) as a control. (middle) Fractions 1–12 correspond with molecular masses from ∼180 to 30 kD. (bottom) Intensity of MBP-Mmr1 bands. Representative of two independent experiments. Black boxes show fractions on SDS-PAGE that correspond to the MBP-Mmr1 monomer peak of ∼50 kD shown in the chromatogram. Abs, absorbance.

Binding site for the post-Golgi Rab GTPases on Myo2 partially overlaps with the Ypt11, Kar9, and Inp2 binding sites. (A, left) Myo2 CBD interacts with Vac17, Mmr1, Kar9, Ypt11, Inp2, and Smy1 in a yeast two-hybrid test. Effects of L1301P are likely caused by conformational changes in the Myo2 CBD. (right) myo2-L1301P, helix 6, affects binding of Vac17, Mmr1, Kar9, and Smy1. Other mutations in this region do not affect Kar9 and Smy1. (B) Surface residues on Myo2 CBD critical for Kar9 and/or Ypt11 binding. (A and B) Incubated at 24°C for 4 d. Top left squares show empty vector controls. (C) Surface (left) and ribbon (right) view of Myo2 CBD indicating surface residues that interact with Kar9, Inp2, Ypt11, and Ypt31/32. Black outline shows Ypt31/32 and Sec4 binding sites. Red: Inp2 only; orange: Kar9 and Inp2; yellow: Kar9 only; green: Kar9, Sec4, and Ypt31/32; blue: Sec4, Ypt11, and Ypt31/32; brown: Kar9, Inp2, Sec4, Ypt11, and Ypt31/32. (D) Myo2 residues L1331, F1334, W1407, K1408, Y1415, and Y1483, the Kar9 binding site, are critical for orientation of the mitotic spindle. G1248D, an internal residue, also affected spindle orientation. myo2Δ cells with wild-type MYO2 or myo2 mutant plasmids transformed with pGFP-Tub1. Orientation of spindle microtubules scored in five categories: (I) Microtubules aligned toward the bud; (II) microtubules improperly aligned away from the mother–bud neck; categories III, IV, and V (Fig. S3) were not informative. Categories I and II were plotted as a percentage of total cells in all five categories. n = 2; ≥215 cells scored per strain. (E) Steady-state levels of Myo2 unchanged in plasmid-containing strains with myo2 point mutations. (F) Myo2 mutants that affect Kar9 and Inp2 interactions had no effect on growth. Mutations in the Rab GTPase binding site affect growth. Strains were grown to log phase, serially diluted, plated on rich media, incubated at 24°C or 37°C for 2–4 d. Bar, 5 µm. AD, activation domain; BD, binding domain.

Model for competition between Vac17 and Mmr1 for access to Myo2. (A) Myo2 transports vacuoles (V) and mitochondria (M). Mmr1/Myo2 localizes to the leading portion of the mitochondrial tubule in the mother near the mother–bud neck and on mitochondria in the bud. Mitochondria move across the mother–bud neck and retract into the mother several times then are stably anchored in the bud. Vac17/Myo2 localizes to the leading portion of vacuoles before entering the bud and on vacuoles in the bud. Vacuoles move across the mother–bud neck and retract into the mother several times and then are anchored in the bud. (B) All cargo-binding regions may be interconnected via conformational changes that modulate the ability of Myo2 to interact with each cargo adaptor. Myo2 CBD showing binding sites for Vac17/Mmr1, Rab GTPase/Inp2/Kar9, and Sec15. Three loops that connect these regions (pink) are labeled F, H, or L according to the 14 loops that appear in the CBD structure, from A to N, beginning at the N terminus. Vac17/Mmr1 binding site was predominantly on helix 6. Myo2-L1331 (lime green) lies within the Rab binding site at the C terminus of helix 6, in loop F. Two loops connect the Sec15 binding site with the Rab GTPase/Inp2/Kar9 binding region and extend from helix 8 (loop H) and helix 12 (loop L) to the Rab/Inp2/Kar9 region.