1. Volume 12, Issue 5, Pages 739-750 (May 2007)
The CORVET Tethering Complex Interacts with the Yeast Rab5 Homolog Vps21 and Is Involved in Endo-Lysosomal Biogenesis 
Karolina Peplowska, Daniel F. Markgraf, Clemens W. Ostrowicz, Gert Bange, Christian Ungermann 
Developmental Cell 
Volume 12, Issue 5, Pages (May 2007)
DOI: /j.devcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Characterization of the vps3Δ and vps8Δ Mutants
(A) Localization of vacuolar protein markers in vps3Δ cells. Wt and vps3Δ cells expressing GFP-tagged vacuolar proteins were grown logarithmically in YPD medium, harvested, washed once with PBS buffer, and analyzed by fluorescence microscopy. Size bar = 5 μm.
(B) Double mutant of vps8 and vam3. Cells lacking Vps8, Vam3, or both were stained with FM4-64 and analyzed by fluorescence microscopy. Size bar = 10 μm.
(C) Colocalization of Vps21 and Ypt7. RFP-Ypt7 and GFP-Vps21 were expressed in wt and vps8Δ cells and visualized by fluorescence microscopy. Images were processed by deconvolution using the Autoquant software. Size bar = 10 μm.
(D) Vacuole inheritance. Wt and vps3Δ cells were logarithmically grown in YPD medium, stained with FM4-64 for 30 min, reisolated, and grown in fresh YPD medium for 3 hr before being analyzed by fluorescence microscopy. Quantification of at least 200 cells of each strain is shown.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2 Identification of the Vps3-Vps8-Class C Complex
(A) Vps3-TAP purification. BJ3505 cells (wt and VPS3-TAP) were lysed and the Vps3-TAP protein was purified according to the TAP protocol (see Experimental Procedures). The eluate was loaded onto a 4%–12% SDS-PAGE gradient gel and candidate proteins were identified by mass spectroscopy. Vps3-cbp (CaM-binding peptide) is left after TEV cleavage.
(B–D) Sizing of Vps3-8-class C complex by gel filtration (B). BY4741 cells carrying Vps3-TAP were lysed and Vps3-TAP was captured on IgG-Sepharose. After TEV cleavage, the eluate was applied onto a Superose 6 column, and proteins in fractions 6–19 were analyzed on a 4%–12% SDS-PAGE gradient gel, followed by Coomassie staining. Bands were identified by mass spectrometry. The same purification from BY4741 VPS8-TAP cells is shown in (C). As a comparison, Vps41-TAP was purified by the same approach (D). Models of the complexes are shown. Class C = Vps11, Vps16, Vps18, Vps33; 8 = Vps8; 3 = Vps3; 41 = Vps41; V6 = Vam6.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3 Interaction of the Rab-GTPase Vps21 with CORVET and Vps3
(A) Interaction of CORVET with Vps21. CORVET was purified from a strain overexpressing Vps3, similar to Figure 2B, and the complex fraction (11, 12) was applied to immobilized GST-Vps21, Ypt1, and Ypt7, which were preloaded with the indicated nucleotide (NF, nucleotide-free). Bound protein was eluted by EDTA/high salt, TCA precipitated, and analyzed by SDS-PAGE and western blotting using Protein A-peroxidase. The Rab-GTPase was eluted by boiling beads in sample buffer and analyzed as above. Note that purified CORVET complex lacks most Vps3 due to its dissociation during chromatography (Figure 2B).
(B and C) Interaction of Vps3 with Vps21. Detergent lysate prepared from 3 l of cells overexpressing Vps3 in the presence (B) or absence (C) of Vps8 was applied to GST-Rabs containing the indicated nucleotide. Analysis was done as in (A).
(D and E) Localization of GFP-Vps21. Cells lacking or overexpressing Vps8, Vps3, or Vam6 in the presence of GFP-Vps21 were analyzed by fluorescence microscopy as in Figure 1A. Size bar = 10 μm.
Developmental Cell  , DOI: ( /j.devcel )
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5. Figure 4 Identification of Two Intermediate Complexes
(A and B) Identification of the Vam6-Vps8-class C intermediate. Cells containing TAP-tagged Vam6 and HA-tagged Vps8 were processed for HOPS purification as in Figure 2B. Proteins in each fraction were resolved on gradient SDS-PAGE gels, which were either stained with Coomassie ([A], 80% of the sample) or transferred to nitrocellulose ([B], 20%). Western blots were first decorated with antibodies to the HA-tag and a mouse secondary antibody coupled to AlexaFluor680 (Molecular Probes; [B], bottom panel), then with antibodies against Vps41 coupled to a goat anti-rabbit secondary antibody coupled to IRDye800 (LI-COR). To visualize both proteins, the channels were merged (top panel). A model of the Vps8-Vam6-class C (i-HOPS) complex is shown.
(C and D) Identification and dynamics of Vps3-Vps41 complexes. Wt or deletion strains containing Vps41-TAP (C) or Vps3-TAP (D) were lysed. Tagged proteins were purified on CaM beads using 700 μg of total protein (see Experimental Procedures). EGTA eluates were TCA precipitated, and proteins were analyzed on a 7.5% SDS-PAGE gel, followed by western blotting with antibodies against the tag, Vps3 or Vps41, Vps33, and Vac8. Note that both the Vps33 and the Vps41 antibodies cross-react in the total lysate with unknown proteins at their apparent molecular weight. The proteins are, however, not detected upon complex purification in the vps33Δ or vps41Δ background, confirming that they are not Vps33 and Vps41, respectively.
(E) Purification and sizing of the Vps3-Vps41-class C complex. TAP-tagged Vps3 was purified from vps8 cells as in Figure 2B. The proteins were analyzed as in (A). The identity of Vps41 was confirmed by western blotting and mass spectrometry (data not shown). The model depicts the Vps3-Vps41-class C (i-CORVET) complex.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5
Effect of Vps3 and Vps8 Overproduction on Complex Composition and Vacuole Morphology
(A) Overproduction of Vps3 leads to severe vacuole fragmentation, whereas overproduction of Vps8 does not influence vacuole morphology. BY VPS41-TAP cells with or without VPS3 or VPS8 under the control of the GAL1 promoter were grown overnight in YPD (glucose) or YPG (galactose) medium, then diluted and grown to logarithmic phase. Cells were stained with FM4-64 and analyzed by fluorescence microscopy (see Experimental Procedures section for the details). Size bar = 10 μm.
(B) Overproduced Vps3 binds to Vps41. BY VPS41-TAP cells with or without VPS3 under the control of the GAL1 promoter were grown overnight in YPD or YPG medium. In the morning, cells were lysed and Vps41-TAP protein was purified as described in Figure 4C and the Experimental Procedures section. Western blots were decorated with antibodies to Vam6, Vps33, Vps3, and Vac8 (as a negative control). A model indicates the conversion of the HOPS complex into the Vps3-Vps41-class C intermediate.
(C) Loss of the Vps3-Vps41-class C complex by overproduction of Vps8. BY wild-type or vam6Δ cells containing VPS41-TAP and VPS8 under the control of the GAL1 promoter were grown overnight in YPD or YPG medium. In the morning, cells were lysed and the Vps41-TAP protein was purified according to the protocol described in the Experimental Procedures section and Figure 4. Proteins copurifying with Vps8 were analyzed by SDS-PAGE and western blotting using antibodies against Vps33 and Vps3. A model indicates the conversion triggered by Vps8 overproduction.
(D) Vacuole morphology in the presence of overproduced Vps3. Diploid cells carrying one allele of VPS3 under the control of the GAL1 promoter were grown in YPD. Synthesis of Vps3 was induced by exchanging the medium for YPG. Cells were incubated with FM hr prior to each time point and incubated for 30 min in YPD (time points 0–1 hr) or YPG (all other time points) medium (pulse), then washed and incubated for an additional 30 min in YPD or YPG (chase). Cells were analyzed by fluorescence microscopy.
(E) Expression control of Vps3. At the same time point of microscopic analysis, an aliquot was processed for whole protein analysis. All samples were analyzed by SDS-PAGE and western blotting using the Protein A-peroxidase (PAP) and the Vps3 antibody.
(F) Pho8 and Ape1 maturation in GAL1-VPS3 strains. Total cell extracts from pep4Δ, wt, and GAL1-VPS3 cells grown in galactose were prepared and analyzed by SDS-PAGE and western blotting using antibodies to Ape1 and Pho8 (m = mature, pro = precursor).
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

7. Figure 6 Directionality of Complex Assembly
(A) Induction and repression of Vps8 expression. Cells with TAP-tagged VPS41 and HA-tagged VPS8 under the control of the GAL1 promoter were grown in medium containing raffinose to repress Vps8 expression, then galactose was added (lane 2) for the indicated time. Afterwards, glucose was added and cells were incubated for an additional 6–18 hr. At each time point, an aliquot was removed from the culture. Cells were lysed, proteins were TCA precipitated, and analyzed by SDS-PAGE and western blotting using antibodies against HA and Vac8.
(B) Conversion from i-CORVET to CORVET is unidirectional. The experiment was performed as in (A). Glucose addition was for 6 hr. Cells were lysed using detergent, and Vps41 was purified using CaM beads (see Experimental Procedures for details). A loading control was removed from the extracts and TCA precipitated. Vps41-TAP-associated proteins were analyzed by SDS-PAGE and western blotting using an antibody to Vps3. Vps3 and Vps41 bands were quantified by laser densitometry. Interaction in lane 1 was set to 100%; interactions in lanes 2 and 3 were 24% and 26%, respectively.
(C) Models of tether dynamics between endosome and vacuole. For details see text.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions