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First published online 18 March 2003
doi: 10.1242/jcs.00395

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Bro1 is an endosome-associated protein that functions in the MVB pathway in Saccharomyces cerevisiae

Greg Odorizzi^1,*, David J. Katzmann^2,, Markus Babst^2,, Anjon Audhya² and Scott D. Emr²

¹ Department of Molecular, Cellular and Developmental Biology, University of Colorado, Campus Box 347, Boulder, CO 80309, USA
² Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, Campus Box 0668, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
Present address: Department of Biochemistry and Molecular Biology, 1611 Guggenheim Building, Mayo Clinic, Rochester, MN 55902, USA
Present address: Microgenomics, 5935 Darwin Court, Carlsbad, CA 92008, USA

View larger version (32K): [in a new window]   Fig. 1. His3-CPS is a reporter for mutations that block the MVB pathway. Schematic diagram of His3-CPS sorting in wild-type cells (A) and in mutant cells defective in either MVB cargo selection or MVB vesicle formation (B). (C) The growth of wild-type cells and a representative class E vps mutant, vps4, that have been streaked onto medium lacking supplemental histidine.

View larger version (59K): [in a new window]   Fig. 2. BRO1 is required for sorting CPS via the MVB pathway. (A) Fluorescence microscopic localization of GFP-CPS and FM 4-64. Arrows indicate class E compartments. (B) Newly synthesized CPS was immunoprecipitated from lysates of cells that had been pulse-labeled with [35S]methionine/cysteine for 10 minutes then chased in non-radioactive medium for 0 or 30 minutes. Immunoprecipitates were resolved by SDS-PAGE and examined by fluorography. (C) Immunoprecipitates of CPS were resolved by SDS-PAGE, transferred to nitrocellulose, and examined by western blotting using anti-ubiquitin antibodies. Note that CPS is normally differentially modified by the addition of two or three oligosaccharide moieties and is, therefore, observed in C as a doublet (Spormann et al., 1992); however, in B, the immunoprecipitates were treated with endoglycosidase H in order to facilitate the detection of precursor CPS (pCPS) and mature CPS (mCPS).

View larger version (75K): [in a new window]   Fig. 3. BRO1 is not essential for CPY sorting. (A) Newly synthesized CPY was immunoprecipitated from the intracellular fraction (I) and the extracellular medium (E) from cells that had been pulse-labeled with [35S]methionine/cysteine for 10 minutes, chased in non-radioactive medium for 30 minutes, then converted to spheroplasts. Immunoprecipitates were resolved by SDS-PAGE and examined by fluorography. p2CPY, Golgi-modified precursor CPY; mCPY, mature CPY. (B) Fluorescence microscopic localization of Vps10-GFP and FM 4-64. The arrows indicate class E compartments.

View larger version (57K): [in a new window]   Fig. 4. Bro1 is a soluble cytoplasmic protein that associates with endosomal membranes. (A) Cell lysates were centrifuged at 13,000 g and 100,000 g. The total protein content of the P13, P100 and S100 fractions was examined by western blotting. Alkaline phosphatase (ALP) is a vacuolar membrane protein, and 3-phosphoglycerate kinase (PGK) is a soluble cytoplasmic enzyme. (B) Fluorescence microscopic localization of GFP-Bro1 and FM 4-64. In the schematic diagram shown above the micrographs, the predicted domains of Bro1 are indicated as BOD (`Bro1 domain'), CC (coiled-coil) and PRD (proline-rich domain). The inset in the top row of panels shows cells subjected to a continuous incubation with FM 4-64 in order to demonstrate the colocalization of GFP-Bro1 with FM 4-64-positive endosomal structures (arrows). Arrows in the bottom set of panels indicate class E compartments. (C) The P13 fraction from vps4 cells was loaded at the bottom of a sucrose density gradient and centrifuged to equilibrium. Fractions of the floating material (F), the non-floating material (NF) and the pellet (P) were collected and analyzed by western blotting. (D) Subcellular fractionation and western blotting was performed as described in A using vps4 cells transformed with low-copy plasmids that encode the vps4K179A (pMB24) or vps4E233Q (pMB49) alleles (Babst et al., 1997).

View larger version (64K): [in a new window]   Fig. 5. The subcellular localization of Bro1 requires ESCRT-III components. (A,B) Subcellular fractionation and western blotting was performed as described in the legend to Fig. 4A. (C) The P13 fraction was collected from vps4 cells, then resuspended in buffer containing 1% Triton X-100 and separated by centrifugation at 100,000 g for 1 hour into a soluble fraction (S100) and a pellet fraction (P100). The total protein content of each fraction was resolved by SDS-PAGE and examined by western blotting.

View larger version (70K): [in a new window]   Fig. 6. Localization of GFP-Bro1 in ESCRT-III mutant cells. Fluorescence microscopic localization of GFP-Bro1 and FM 4-64. The arrows indicate GFP-Bro1 localization to class E compartments.