Vacuole-targeted transmembrane proteins accumulate on endosomes and do not reach the vacuolar lumen in certain COPI mutants. (A) Fur4 does not target to the vacuolar lumen in COPIb mutants. Wild-type (WT) yeast (W303-1a) and strains with mutations in COPIf (sec21-1, sec21-2, and ret2-1), COPIb (sec27-1, sec28Δ [RDY241], sec33-1, and ret1-3), ESCRT (vps23Δ), and endocytosis (end4-1) were transformed with a multicopy plasmid expressing FUR4-GFP (pADH-FUR4-GFP) and examined by confocal microscopy. Both phase-contrast microscopy (PC) and merged (MERGE) panels are indicated. (B) Ste2 does not target to the vacuolar lumen in a COPIb mutant. Wild-type yeast (W303-1a) and a COPIb mutant (sec28Δ [RDY241]) were transformed with a single-copy plasmid producing either Ste2-GFP (pRS314-STE2-GFP) or Ste2Δtail-GFP (pRS314-STE2Δtail-GFP; Ste2Δtail). Strains with mutations in COPIf (sec21-1, sec21-2, ret2-1, and ret3-1), COPIb (sec33-1 and ret1-3), ESCRT (vps23Δ), and END4 (end4-1) were transformed with a multicopy plasmid expressing STE2-GFP (pSTE2-GFP). Arrows indicate the position of endosomal compartments adjacent to the vacuole in which GFP-tagged markers accumulate. (C) Ste3 does not target to the vacuolar lumen in COPIb mutants. Wild-type yeast (W303-1a) and strains with mutations in COPIf (sec21-1, sec21-2, ret2-1, and ret3-1), COPIb (sec27-1, sec28Δ [RDY241], sec33-1, and ret1-3), ESCRT (vps23Δ), and END4 (end4-1) were transformed with a single-copy plasmid expressing STE3-GFP (pSTE3-GFP) and examined by confocal microscopy. Arrows indicate the position of endosomal compartments adjacent to the vacuole in which Ste3-GFP accumulates.

FM4-64 labeling of an enlarged late endosome in certain COPI mutants. (A) FM4-64 staining of endocytic membranes in COPIb mutants. Wild-type (WT) cells (W303-1a) and mutants in COPIf (sec21-2), COPIb (sec27-1, sec28Δ, and sec33-1), and ESCRT (vps23Δ) were grown to mid-log phase and subjected to endosomal and vacuolar labeling with FM4-64 to visualize these organelles (see Materials and Methods). Arrows indicate positions of class E compartment in which FM4-64 accumulates. Phase-contrast microscopy (PC) panels are indicated. (B) Colocalization of FM4-64 with accumulated transmembrane proteins in COPIb mutants. sec28Δ (RDY241) and sec27-1 yeast cells expressing GFP-CPS1 or Fur4-GFP proteins were stained with FM4-64 by pulse-chase labeling at 26°C. Both phase-contrast microscopy (PC) and merged (MERGE) panels are indicated.

Sec28 localizes to endosomal compartments labeled by FM4-64 and Vps27. (A) SEC28-GFP and SEC21-GFP, expressed from the genome, partially colocalize with FM4-64. Wild-type (WT) yeast (BY4741) and a class E mutant (vps23Δ) expressing intragenomic SEC28-GFP (Sec28-GFP; GGY3, and GGY6, respectively) and SEC21-GFP (Sec21-GFP; GGY7 and GGY8, respectively) were labeled with FM4-64 and examined by confocal microscopy. Arrows indicate the Sec28-GFP- and Sec21-GFP-labeled endosomal compartments in which FM4-64 accumulates. (B) The upper panels show overexpressed GFP-SEC28 partially colocalizes with FM4-64 labeling of the endocytic pathway. Wild-type yeast cells (BY4741) and class E mutant cells (vps23Δ and vps37Δ) expressing GFP-SEC28 from a multicopy plasmid (pADH-GFP-SEC28) were labeled with FM4-64 and examined by confocal microscopy. Arrows indicate GFP-Sec28-labeled class E compartments in which FM4-64 accumulates. The lower panel shows that GFP-Vps27 and RFP-Sec28 partially colocalize. Wild-type yeast cells (W303-1a) and mutant vps23Δ and vps4Δ cells expressing RFP-SEC28 from a multicopy plasmid (pADH-RFP-SEC28) and GFP-VPS27 expressed from a multicopy plasmid (pGFP-VPS27) were examined by confocal microscopy. An arrow indicates one of the colabeled endosomal compartments in these cells, while arrowheads point out juxtaposed RFP-Sec28- and GFP-Vps27-labeled compartments. Both phase-contrast microscopy (PC) and merged (MERGE) panels are indicated. (C) z sections of cells showing Sec28 colocalized with endosomal markers. The upper panels show class E mutant cells (vps23Δ) expressing GFP-SEC28 from a multicopy plasmid (pADH-GFP-SEC28) were labeled with FM4-64 and examined by confocal microscopy. The lower panels show class E mutant cells (vps23Δ) expressing RFP-SEC28 (pADH-RFP-SEC28) and GFP-VPS27 (pGFP-VPS27) were examined by confocal microscopy. z sections were taken in increments of 0.5 μm. (D) A model for COPIb in endosomal protein sorting in yeast. Endocytosed membrane proteins Snc1 (blue) and Ste2 or Fur4 (red) is first targeted to the early endosome (red and blue circle) by carrier vesicles. Next, sorting of Snc1 to the trans-Golgi apparatus and Fur4 and Ste2 to the late (recycling) endosome occurs. Biosynthetic soluble cargo (i.e., CPY [green]) and membrane cargo (i.e., CPS1 [brown]) are sorted from the trans-Golgi apparatus to the late endosome (large red and brown circle) as well. MVB biogenesis and protein sorting therein are mediated in a COPIb- and ESCRT-dependent manner (see Discussion). During MVB biogenesis, intralumenal vesicles (small brown and red circles) are delivered to the vacuolar lumen where they are degraded. COPI indicates ER-Golgi and intra-Golgi apparatus retrograde transport steps regulated by this coat complex. COPII indicates the ER-Golgi apparatus anterograde transport steps regulated by this coat complex. RET indicates the retromer complex involved in late endosome-trans-Golgi recycling. Recycled Fur4 and, possibly, Ste2 are delivered to the plasma membrane by secretory vesicles derived from late endosomes.

Yeast strains with mutations in COPIb secrete CPY, but not Kar2. (A) Wild-type (WT) yeast (W303-1a) and strains with mutations in COPIf (sec21-2), COPIb (sec27-1, sec28Δ [RDY241], and sec33-1; gifts of R. Duden), ESCRT (vps23Δ, vps28Δ, and vps37Δ), and vam3Δ were patched onto nitrocellulose filters and grown upon solid rich medium for 1 to 2 days at 26°C. Cells secreting CPY and Kar2 were identified by immunodetection (see Materials and Methods). Both the vam3Δ and ESCRT mutants served as positive controls for CPY secretion. (B) Various wild-type cells and COPI mutants do not secrete CPY. Wild-type yeast (BY4741, W303-1a, NY13, and SEY6210) and strains with mutations in COPIb (sec27-1 and ret1-3), COPIf (sec21-1, ret2-1, and ret3-1), and ESCRT (vps23Δ) were patched onto nitrocellulose filters and treated as described in the legend to Fig. 4A. (C) p2-CPY is secreted into the medium from a COPI mutant. Five OD₆₀₀ U of wild-type yeast (BY4741), ESCRT (vps23Δ), and COPIb (sec27-1) was grown in YPD medium containing 50 mM KPO₄, pH 5.7, for 1 h at 30°C. Cultures were harvested by centrifugation to separate the intracellular (I; cells) and extracellular (E; medium) fractions. The fractions were treated as detailed in Materials and Methods, and aliquots of each were separated by SDS-PAGE. The positions of the precursor (p2-CPY) and mature (m-CPY) forms of CPY are indicated. Relative amounts (in percent) of the secreted (p2-CPY) and intracellular (m-CPY) forms of CPY for each strain, as calculated using densitometry, are presented below the Western blot. (D) Growth sensitivity of various COPIb strains. Wild-type yeast (BY4741) and ESCRT (vps23Δ) and COPIb (sec27-b1, sec28Δ #1 [Y01469] and sec28Δ #2 [RDY241]) mutant cells were grown on glucose-containing medium; and yeast strains with a galactose-inducible SEC28 allele (GAL-SEC28 and GAL-SEC28 vps23Δ) were maintained on galactose-containing medium prior to shifting to glucose-containing medium for 20 h. Cells were then diluted serially (10-fold dilutions) and plated onto solid medium. Cells were grown at various temperatures (26°C to 37°C, as indicated) for 2 days. (E) COPIb strains secrete different levels of CPY. The same strains as shown in panel D were grown at 26°C on a nitrocellulose filter on a YPD plate. CPY detection was performed as described for panel A. (F) Suppression of SEC28 expression in glucose-containing media. Wild-type and vps23Δ yeast cells bearing GAL-SEC28 (GGY1 and GGY2, respectively) were grown on either galactose- or glucose-containing medium for 20 h, lysed, and subjected to Western blot analysis using anticoatomer antibodies.

CPS1 localization is impaired, although CPY^1-50GFP localizes normally, in certain COPI mutants. (A) CPS1 does not target to the vacuolar lumen in COPIb mutants. Wild-type (WT) yeast (W303-1a) and strains with mutations in COPIf (sec21-2, ret2-1, and ret3-1), COPIb (sec27-1, sec28Δ [RDY241], sec33-1, and ret1-3), ESCRT (vps23Δ), and RCY1 (rcy1Δ) were transformed with a multicopy plasmid expressing GFP-CPS1 (pGFP-CPS1) and examined by confocal microscopy. Both phase-contrast microscopy (PC) and merged (MERGE) panels are indicated. (B) CPY-GFP localizes to the vacuole in COPIb mutants. Wild-type yeast (W303-1a) and strains with mutations in COPIb (sec27-1 and sec28Δ [RDY241]), COPIf (sec21-1, ret2-1, and ret3-1), ESCRT (vps23Δ), and RCY1 (rcy1Δ) were transformed with a single-copy plasmid expressing CPY^1-50-GFP (pGALΔBglII-CPY[1-50]GFP) and examined by confocal microscopy.

Vps27 binds to subunits of COPIb. (A) Coimmunoprecipitation of GFP-Vps27 with COPIb. Class E vps mutant yeast (vps23Δ) bearing multicopy plasmids expressing myc-SEC27 (pADH-SEC27), myc-SEC28 (pADH-SEC28), or myc-SEC33 (pADH-SEC33), together with a second plasmid expressing GFP-VPS27 (pGFP-VPS27), were lysed and subjected to immunoprecipitation (IP) with anti-myc antibodies. Detection of the precipitated proteins in immunoblots (IB) was performed with anti-Vps27 (1:1,000), anticoatomer (1:1000), or anti-myc (1:1,000) antibodies. “Vector” indicates cells transformed with an empty vector (pAD6). −, cells lacking the GFP-Vps27 protein; +, cells producing the GFP-Vps27 protein. Samples of the TCL are shown (50 μg protein/lane). (B) Sec33 interacts with Vps27 in cells lacking Sec28. Wild-type (WT) yeast (BY4741), a class E vps mutant (vps23Δ), or SEC28-deficient cells (sec28Δ) bearing multicopy plasmids expressing myc-SEC33 (pADH-SEC33) and GFP-VPS27 (pGFP-VPS27) were lysed and subjected to immunoprecipitation as described for panel A. (C) A control protein, Vsm1, does not precipitate myc-Sec27. Wild-type yeast (BY4741) cells bearing multicopy plasmids expressing myc-SEC27 (pADH-SEC27) or myc-VSM1 (pADH-VSM1), together with a second plasmid expressing GFP-VPS27 (pGFP-VPS27), were subjected to immunoprecipitation as described for panel A. Detection of the precipitated proteins in immunoblots was performed with anti-Vps27 (1:1,000) or anti-myc (1:1,000) antibodies. (D) Coimmunoprecipitation of Sec28-GFP with Vps27-myc expressed from the genome. Wild-type yeast (W303-1a) cells bearing integrated SEC28-GFP (GGY3), VPS27-MYC (GGY4), or both (GGY5) were lysed and subjected to immunoprecipitation. −, cells lacking the integration; +, cells bearing the integration of the corresponding tag. Detection of the precipitated proteins in immunoblots was performed with anti-GFP (1:1,000) or anti-myc (1:1,000) antibodies. Samples of the TCL are shown (50 μg protein/lane).