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First published online October 11, 2005
doi: 10.1242/10.1242/jcs.02599

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Yeast Mon2p is a highly conserved protein that functions in the cytoplasm-to-vacuole transport pathway and is required for Golgi homeostasis

Jem A. Efe¹, Fabienne Plattner², Nicolas Hulo³, Dieter Kressler^2,*, Scott D. Emr¹ and Olivier Deloche^2,

¹ Division of Biology, Department of Cellular and Molecular Medicine, and the Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 920930668, USA
² Département de Microbiologie et Médecine Moléculaire, Centre Médical Universitaire, Université de Genève, Genève, Switzerland
³ Institut Suisse de Bioinformatique, Centre Médical Universitaire, Université de Genève, Genève, Switzerland

View larger version (25K): [in a new window]   Fig. 1. Identification of a Mon2p protein family. (A) Representation of six conserved domains (A to F) present in Mon2p and in nine other uncharacterized proteins. Blue boxes are domains with significant levels of sequence similarity, which were only detected in Mon2p and the identified proteins. Green boxes represent domains that are also present in all members of the large Sec7 protein family. The dark-green bar in domains C and HUS is a Y-D motif detected in the conserved region described previously (Jackson and Casanova, 2000). The percentage (%) similarity of each domain is indicated. (B) Representation of Sec7p (S. cerevisiae) and BIG1 (H. sapiens), two members of the large Sec7 protein family, with their Sec7 catalytic domain represented by orange boxes. Recently, the domain architecture of the Sec7/BIG1 protein family has been redefined (Mouratou et al., 2005), and this alternative configuration is depicted below the dashed line for comparison. The first two conserved DCB (dimerization/cyclophilin binding) and HUS (homology upstream of Sec7) domains correspond to our B and C domains respectively, whereas the three HDS1, HDS2 and HDS3 (homology downstream of Sec7) regions overlap with our D domain. All protein sequences were taken from the Swiss-Prot/EMBL databases. S.c., Saccharomyces cerevisiae; H.s., Homo sapiens; M.m., Mus musculus; N.c., Neurospora crassa; As.g., Ashbya gossypii; S.p., Schizosaccharomyces pombe; C.e., Caenorhabditis elegans; An.g., Anopheles gambiae; D.m., Drosophila melanogaster; O.s., Oryza sativa.

View larger version (34K): [in a new window]   Fig. 2. The C-terminal region of Mon2p is essential for cell growth under membrane stress. (A) Schematic representation of the Mon2p-GFP fusion with its six conserved domains (A-F) and various truncated Mon2p-GFP derivatives: Mon2[A-F]p/(pFP45), Mon2[A-E]p/(pOD123), Mon2[A-d]p/(pFP46), Mon2[A-C]p/(pOD124), Mon2[A-B]p/(pOD125), Mon2[A]p/(pFP46), Mon2[B-F]p/(pOD126), Mon2[d-F]p/(pOD122) and Mon2[E-F]p/(pOD127). The D domain is denoted with a lowercase d when it is not entirely expressed. Growth complementation of the different Mon2p-GFP derivatives were tested in YFP44 (mon2/chc1-521) cells at 30°C and indicated as followed: +++, growth identical to wild-type cells; ++ and +, growth is slightly and strongly reduced, respectively; –, no growth. (B) Western blotting. Whole cell extracts of YFP93 (mon2) cells transformed with constructs encoding GFP-tagged truncations (lanes labelled 1-9 correspond to constructs in A) were resolved by SDS-PAGE. The expressed protein fragments were detected by GFP antibody. A released GFP fragment is labeled with 1 and the presence of a doublet with 2. (C) YFP93 (mon2) cells expressing the Mon2p-GFP derivatives containing the indicated domains were grown on SD-LEU plates containing 0 or 20 µM CPZ for 36 hours at 25°C.

View larger version (55K): [in a new window]   Fig. 3. Mon2p is associated with the late Golgi and early endosomes. (A) Mon2p-GFP colocalizes with Kex2p-HA and Pep12p on a sucrose density gradient. JEY003 (MON2-GFP) cells harboring pRS316-KEX2-HA were grown to mid-logarithmic phase at 30°C and subsequently lysed. The P100 fraction was prepared and subjected to equilibrium sedimentation through a sucrose gradient. Fractions were collected from the top, subjected to SDS-PAGE and analyzed by western blotting using anti-GFP, anti-HA, anti-Mnn1p and antiPep12p antibodies. (B) Mon2p-GFP partially colocalizes with late-Golgi markers (Sec7pDsRed and PHFAPP1-DsRed), but shows no overlap with a late endosomal marker (DsRedFYVEEEA1). Strains chromosomally expressing Mon2p-GFP and harboring the indicated fusion construct on a plasmid were grown to mid-logarithmic phase and observed by fluorescence microscopy as described in Materials and Methods. Images are representative of more than 100 cells observed.

View larger version (67K): [in a new window]   Fig. 4. The N-terminal region binds to the late Golgi/endosomal membranes. (A) YFP93 (mon2) cells expressing Mon2[A-F]p-GFP/(pFP45), Mon2[A-E]p-GFP/(pOD123), Mon2[A-B]p-GFP/(pOD125), Mon2[A]p-GFP/(pFP46), Mon2[d-F]p-GFP/(pOD122), Mon2[A-C]p/(pOD124) and Mon2[A-d]p/(pFP46) proteins were grown to mid-logarithmic phase at 30°C and processed for fluorescence microscopy. MON2[A-d] was subcloned into a centromeric (CEN) plasmid (pOD130) to reduce protein expression level and the fluorescence was detected with a confocal microscope (Nipkow). The observed localization pattern is indicated below each image. (B) For sucrose density gradients, YFP93 cells transformed with pRS316KEX2-HA and pFP45, YFP93 cells transformed with pOD123, pOD124, pOD126 or pOD127 and ODY318 (arl1::kanMX4) cells containing pFP45 were grown to mid-logarithmic phase at 30°C and subjected to equilibrium sedimentation through a sucrose gradient. Fractions were collected from the top and analyzed by western blotting using anti-HA, anti-Pep12p and anti-GFP antibodies. The first three proteins, indicated by a vertical bar, were detected from the same sucrose gradient.

View larger version (45K): [in a new window]   Fig. 5. The transport of the prApe1 to the vacuole is blocked in the mon2 mutant. (A) YFP95 (wild type) and YFP93 (mon2) were metabolically (35S) labeled at 30°C for 10 minutes and chased for the indicated times. The ER, Golgi and mature forms of CPY are labeled p1, p2 and m, respectively. (B) SEY6210 (wild type) and AAY1120 (mon2) cells were metabolically (35S) labeled at 26°C for 20 minutes and chased for the indicated times. CPY (A) and Ape1 (B) were immunoprecipitated from extracts and analyzed by SDS-PAGE and autoradiography. (C) Subcellular localization of GFP-prApe1. SEY6210 and AAY1120 cells expressing GFP-prApe1 were grown at 26°C to mid-logarithmic phase and labelled with FM4-64 to visualize vacuoles. Analysis was by fluorescence microscopy and images shown are representative of more than 100 cells observed.

View larger version (153K): [in a new window]   Fig. 6. EM analysis of subcellular structure. SEY6210 (wild-type) (A), AAY1120 (mon2) cells grown at 26°C (B,C,D) and shifted 38°C for 2 hours (F,H), and AAY104 (pik1ts) cells pre-shifted to the restrictive temperature for one hour (E,G) were processed for and visualized by EM as described in Materials and Methods. Black arrowheads indicate enlarged aberrant membrane structures shown enlarged in the panels on the right. Vacuoles (v), nuclei (n) and mitochondria (m) are also indicated. Cells shown are representative of >90% of the population (except for multinucleation, which was observed in 15% of Dmon2 cells). Bar, 0.5 µm.

View larger version (31K): [in a new window]   Fig. 7. Mon2p associates with large protein complexes and forms oligomers. (A) Native lysate from JEY001 cells (Mon2p-HA; 100 OD600 units) was cleared by ultracentrifugation at 100,000 g for 1 hour and subjected to gel filtration chromatography. Fractions were analyzed by SDS-PAGE and western blotting using a monoclonal anti-HA antibody. All fractions containing Mon2p-HA are shown and approximate complex size (based on a prior run of sizing standards) is indicated. (B) Mon2p-myc coimmunoprecipitates with Mon2p-HA. Detergent-solubilized native lysates from cells (50 OD600 units) expressing Mon2p-myc alone (JEY003) or with Mon2p-HA (JEY026) were cleared at 16,000 g and subjected to immunoprecipitation using anti-HA antibody. Purified proteins were subjected to SDS-PAGE and western blotting analysis with anti-myc and anti-HA antibodies.

View larger version (79K): [in a new window]   Fig. 8. Isolation of ARL1, NEO1 and DOP1 as multicopy suppressors of the mon2 mutant. ARL1, NEO1 and DOP1 were isolated in CDK13-1A (mon2/tif3) double mutant as described in Materials and Methods. The complementation of the mon2 mutant was subsequently tested in YFP44 (mon2/chc1-521ts) double mutant. YFP44 cells were transformed with plasmids (2 µ, and the appropriate auxotrophic markers) alone or plasmids containing MON2 (pFP45), DOP1 (pDK64), NEO1 (pRB111) or ARL1 (pRB95). The resulting transformants were then tested for growth at 30°C on 5-FOA-containing plates as indicated. No growth complementation was observed in YFP86 (tif3/vps54) double mutant (data not shown), which was used as a control.

View larger version (53K): [in a new window]   Fig. 9. Mon2p associates with the peripheral membrane protein Dop1p. (A) Dop1p-HA-mRFP is peripherally associated with membranes. JEY069 (MON2-GFP DOP1-HA-mRFP) cells (20 OD600 units) were osmotically lysed and centrifuged at 13,000 g. The supernatant (S13) fraction was differentially treated as indicated, and further separated by ultracentrifugation at 100,000 g. The resulting high-speed pellet (P) and supernatant (S) fractions were analyzed for relative Dop1p-HA-mRFP content by western blotting. (B) Mon2p colocalizes with Dop1p. JEY069 cells were grown to mid-logarithmic phase at 26°C, shifted to 4°C for 10 minutes to minimize rapid movement of Golgi and endosomal elements, and observed by fluorescence microscopy. (C) Mon2p-GFP coimmunoprecipitates with Dop1p-HA-mRFP. Detergent-solubilized native lysates (50 OD600 units) from JEY003 (MON2-GFP) and JEY069 were cleared at 16,000 g and subjected to immunoprecipitation using anti-HA antibody. Purified proteins were subjected to SDS-PAGE and western blotting analysis with anti-GFP and anti-HA antibodies. * indicates a possible breakdown product of Dop1p-HA-mRFP.

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