-
1
-
-
0033130103
-
-
S. R. Pfeffer, Nature Cell Biol. 1, 17 (1999); P. Novick and M. Zerial, Curr. Opin. Cell Biol. 9, 496 (1997); A. Mayer, ibid., in press.
-
(1999)
Nature Cell Biol.
, vol.1
, pp. 17
-
-
Pfeffer, S.R.1
-
2
-
-
0030860083
-
-
S. R. Pfeffer, Nature Cell Biol. 1, 17 (1999); P. Novick and M. Zerial, Curr. Opin. Cell Biol. 9, 496 (1997); A. Mayer, ibid., in press.
-
(1997)
Curr. Opin. Cell Biol.
, vol.9
, pp. 496
-
-
Novick, P.1
Zerial, M.2
-
3
-
-
0033130103
-
-
in press
-
S. R. Pfeffer, Nature Cell Biol. 1, 17 (1999); P. Novick and M. Zerial, Curr. Opin. Cell Biol. 9, 496 (1997); A. Mayer, ibid., in press.
-
Curr. Opin. Cell Biol.
-
-
Mayer, A.1
-
5
-
-
0029980441
-
-
A. Mayer, W. Wickner, A. Haas, Cell 85, 83 (1996); B. Conradt, A. Haas, W. Wickner, J. Cell Biol. 126, 99 (1994).
-
(1996)
Cell
, vol.85
, pp. 83
-
-
Mayer, A.1
Wickner, W.2
Haas, A.3
-
6
-
-
0028225582
-
-
A. Mayer, W. Wickner, A. Haas, Cell 85, 83 (1996); B. Conradt, A. Haas, W. Wickner, J. Cell Biol. 126, 99 (1994).
-
(1994)
J. Cell Biol.
, vol.126
, pp. 99
-
-
Conradt, B.1
Haas, A.2
Wickner, W.3
-
7
-
-
0345344454
-
-
data not shown
-
C. Peters et al., data not shown.
-
-
-
Peters, C.1
-
8
-
-
0344050665
-
-
note
-
2, 40 mM CHAPS, 75 μM pefablock SC, 150 nM leupeptin, 37.5 μM o-phenanthroline, 500 nM pepstatin A) and centrifuged (100,000g, 30 min, 4°C). The supernatant was passed over 100 μl of microcystin LR-Sepharose (6) (200 μl/min). The matrix was washed with 25 ml of buffer A and eluted with 1 ml of buffer A containing 50 μM microcystin LR. Eluted proteins were concentrated by ultrafiltration in microcon-10 (Amicon).
-
-
-
-
9
-
-
0028132006
-
-
G. Moorhead, R. W. Mackintosh, N. Morrice, T. Gallagher, C. Mackintosh, FEBS Lett. 356, 46 (1994); R. W. MacKintosh et al., ibid. 371, 236 (1995).
-
(1994)
FEBS Lett.
, vol.356
, pp. 46
-
-
Moorhead, G.1
Mackintosh, R.W.2
Morrice, N.3
Gallagher, T.4
Mackintosh, C.5
-
10
-
-
0029088182
-
-
G. Moorhead, R. W. Mackintosh, N. Morrice, T. Gallagher, C. Mackintosh, FEBS Lett. 356, 46 (1994); R. W. MacKintosh et al., ibid. 371, 236 (1995).
-
(1995)
FEBS Lett.
, vol.371
, pp. 236
-
-
MacKintosh, R.W.1
-
12
-
-
0029927505
-
-
A. Shevchenko, M. Wilm, O. Vorm, M. Mann, Anal. Chem. 68, 850 (1996).
-
(1996)
Anal. Chem.
, vol.68
, pp. 850
-
-
Shevchenko, A.1
Wilm, M.2
Vorm, O.3
Mann, M.4
-
13
-
-
10544244161
-
-
A. Shevchenko et al., Proc. Natl. Acad. Sci. U.S.A. 93, 14440 (1996); O. N. Jensen, A Podtelejnikov, M. Mann, Rapid Commun. Mass Spectrom. 10, 1371 (1996).
-
(1996)
Proc. Natl. Acad. Sci. U.S.A.
, vol.93
, pp. 14440
-
-
Shevchenko, A.1
-
14
-
-
0029808148
-
-
A. Shevchenko et al., Proc. Natl. Acad. Sci. U.S.A. 93, 14440 (1996); O. N. Jensen, A Podtelejnikov, M. Mann, Rapid Commun. Mass Spectrom. 10, 1371 (1996).
-
(1996)
Rapid Commun. Mass Spectrom.
, vol.10
, pp. 1371
-
-
Jensen, O.N.1
Podtelejnikov, A.2
Mann, M.3
-
15
-
-
0344050664
-
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note
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YER133w is the GLC7 gene. The peptides matched within 20 parts per million and covered 33% of the Glc7p sequence. For mass spectrometry, protein bands were excised from the gels and digested by trypsin (8). We analyzed 2% of the supernatant by MALDI mass spectrometry on a Bruker REFLEX instrument (Bruder Daltonics, Bremen, Germany) and searched the resulting peptide mass maps in a non-redundant sequence database using PeptideSearch software developed by P. Mortensen and M. Mann. Hits were verified with second-pass searches taking account of chemical modifications.
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-
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0345344451
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note
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Standard fusion yeast strains were BJ3505 and DKY6281 (3). One unit of fusion activity is defined as 1 μmol of p-nitrophenol developed per minute and microgram of Δpep4 vacuoles. Vacuole isolation, cytosol preparation, and fusion were performed and assayed as described (7); however, the cells for the cytosol preparation were not lysed in a bead beater but by freezing the cell suspension and blending it in liquid nitrogen. The cytosol concentration in fusion assays was only 0.6 mg/ml. Anti-PP1 was raised by blotting PP1 from an SDS gel onto nitrocellulose, dissolving the nitrocellulose in dimethyl sulfoxide, and subcutaneously injecting a 1:1 mixture with Freund's adjuvant into rabbits. Anti-PP1 was affinity purified with a PP1-glutathione S-transferase (GST) fusion protein immobilized on CH-Sepharose 4B (Pharmacia) (3). However, after the coupling procedure the column was washed with 6 M guanidinium-HCl, 10 mM EDTA to denature the protein. This was critical for successful extraction of inhibitory antibodies.
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0345344450
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note
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FPLC column (Pharmacia; 0.2 ml/min). The column was washed with 10 ml of 50 mM tris pH 8, 50 mM NaCl. Bound proteins were eluted with an NaCl gradient (100 mM to 1.5 M). Fractions containing inhibitor 2 (determined by protein immunoblot) were pooled, and the buffer was changed to 10 mM Pipes-KOH pH 6.8, 200 mM sorbitol, 150 mM KCl by repeated concentration and dilution in a centricon-10. Samples were stored at -20°C.
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19
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0344913186
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note
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2, 50% glycerol by repeated concentration and dilution in a centricon-10 and stored at -80°C.
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-
-
-
20
-
-
0030451563
-
-
M. J. R. Stark, Yeast 12, 1647 (1996).
-
(1996)
Yeast
, vol.12
, pp. 1647
-
-
Stark, M.J.R.1
-
21
-
-
0344913187
-
-
note
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+) were generated by crossing this strain with PAY700-4 and PAY704-1, respectively, then selecting haploid segregants with the appropriate combination of genetic markers after tetrad analysis. PAY821-2Bα (MATα glc7::LEU2 trp1:: glc7-10::TRP1 pep4Δ::URA3) and PAY820-1Dα (MATα glc7::LEU2 trp1::GLC7::TRP1 pep4Δ::URA3) were similarly generated after crossing WZY333 (MATαpep4Δ:: URA3) with PAY700-4 and PAY704-1 and additionally verified by using a standard plate assay to confirm the lack of CPY activity after replating several times (31).
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0344050663
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note
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Gdi1p extracts the Rab-like GTPase Ypt7p from the membrane and prevents docking (7). Anti-PP1 was not as effective in this type of experiment as in Fig. 1B because the antibody binds slowly to PP1 on the membrane (4). Because the kinetic analysis in Fig. 3 did not allow a preincubation of vacuoles with the antibodies as in Fig. 1B, the inhibitory effect of the antibodies was not complete, shifting the inhibition curve to the left. Microcystin LR binds covalently to PP1, resulting in rapid inactivation.
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-
-
-
24
-
-
0031047480
-
-
Z. Xu, A. Mayer, E. Muller, W. Wickner, J. Cell Biol. 136, 299 (1997); Z. Xu, K. Sato and W. Wickner, Cell 93, 1125 (1998).
-
(1997)
J. Cell Biol.
, vol.136
, pp. 299
-
-
Xu, Z.1
Mayer, A.2
Muller, E.3
Wickner, W.4
-
25
-
-
0032568798
-
-
Z. Xu, A. Mayer, E. Muller, W. Wickner, J. Cell Biol. 136, 299 (1997); Z. Xu, K. Sato and W. Wickner, Cell 93, 1125 (1998).
-
(1998)
Cell
, vol.93
, pp. 1125
-
-
Xu, Z.1
Sato, K.2
Wickner, W.3
-
26
-
-
0344913183
-
-
note
-
3, and 1 mM PMSF for 5 min at 95°C. The SDS concentration was adjusted to 0.2% by dilution with IP buffer (50 mM tris pH 7.4, 0.2% Tween 20, 150 mM KCl. 100 μM EDTA, 1 mM PMSF). CPY was immunoprecipitated by adding affinity-purified antibodies (2 μg/ml) and 50 μl of a 1:1 slurry of protein A-Sepharose to 1 ml of lysate. After 2 hours of shaking at 4°C, the beads were pelleted and washed three times with IP buffer and once with phosphate-buffered saline. Proteins were eluted with SDS sample buffer.
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-
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-
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-
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0028929242
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This dye rapidly associates with the plasma membrane, becomes endocytosed, and migrates by vesicular transport to the vacuole where it accumulates [T. Vida and S. D. Emr, J. Cell Biol. 128, 779 (1995)].
-
(1995)
J. Cell Biol.
, vol.128
, pp. 779
-
-
Vida, T.1
Emr, S.D.2
-
29
-
-
0345344447
-
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note
-
600 = 10. A 7-μl sample of the suspension was mixed with 7 μl of 0.4% Seaplaque agarose in 10 mM Pipes-KOH pH 6.8, 200 mM sorbitol (kept liquid at 35°C), transferred to a microscopy slide, and chilled at 4°C for 5 min to immobilize the cells. Cells were quickly investigated with a confocal microscope (Lelca TCS) under minimal excitation.
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0345344442
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note
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FPLC column (Pharmacia) equilibrated in buffer S with 4 mM CHAPS. The column was run at 0.5 ml/min (4°C). We collected 1-ml fractions.
-
-
-
-
31
-
-
0032549708
-
-
T. Weber et al., Cell 92, 759 (1998); M. Otter-Nilsson, R. Hendriks, E.-I. Pecheur-Huet, D. Hoekstra, T. Nilsson, EMBO J. 18, 2074 (1999).
-
(1998)
Cell
, vol.92
, pp. 759
-
-
Weber, T.1
-
32
-
-
0033560750
-
-
T. Weber et al., Cell 92, 759 (1998); M. Otter-Nilsson, R. Hendriks, E.-I. Pecheur-Huet, D. Hoekstra, T. Nilsson, EMBO J. 18, 2074 (1999).
-
(1999)
EMBO J.
, vol.18
, pp. 2074
-
-
Otter-Nilsson, M.1
Hendriks, R.2
Pecheur-Huet, E.-I.3
Hoekstra, D.4
Nilsson, T.5
-
33
-
-
0032506543
-
-
C. Ungermann, K. Sato, W. Wickner, Nature 396, 543 (1998); J. R. Coorssen, P. S. Blank, M. Tahara, J. Zimmerberg, J. Cell Biol. 143, 1845 (1998).
-
(1998)
Nature
, vol.396
, pp. 543
-
-
Ungermann, C.1
Sato, K.2
Wickner, W.3
-
34
-
-
0032576575
-
-
C. Ungermann, K. Sato, W. Wickner, Nature 396, 543 (1998); J. R. Coorssen, P. S. Blank, M. Tahara, J. Zimmerberg, J. Cell Biol. 143, 1845 (1998).
-
(1998)
J. Cell Biol.
, vol.143
, pp. 1845
-
-
Coorssen, J.R.1
Blank, P.S.2
Tahara, M.3
Zimmerberg, J.4
-
35
-
-
0030968878
-
-
B. J. Nichols, C. Ungermann, H. R. B. Pelham, W. T. Wickner, A. Haas, Nature 387, 199 (1997).
-
(1997)
Nature
, vol.387
, pp. 199
-
-
Nichols, B.J.1
Ungermann, C.2
Pelham, H.R.B.3
Wickner, W.T.4
Haas, A.5
-
38
-
-
0031820288
-
-
M. S. Longtine et al., Yeast 14, 953 (1998).
-
(1998)
Yeast
, vol.14
, pp. 953
-
-
Longtine, M.S.1
-
41
-
-
0344050656
-
-
note
-
We thank C. Macintosh for helpful hints in the initial phase of the project, K. Nasmyth for strain WZY333, M. Bayer and O. Müller for discussions, and E. Schlegel for help in preparing the manuscript. Supported by the Wellcome Trust (project grants 046956 and 049778, M.J.R.S.), the Deutsche Forschungsgemeinschaft (grant SFB445, A.M.), and the Boehringer Ingelheim Stiftung (A.M.).
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