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1.
Figure 5

Figure 5. From: Inositol Phosphorylceramide Synthase Is Located in the Golgi Apparatus of Saccharomyces cerevisiae.

Aur1p-HA cofractionates with IPC synthase activity. Membranes prepared from strain SEY6210 with endogenous Aur1p tagged with HA were fractionated on a velocity gradient and assayed for IPC synthase activity or for the indicated proteins by quantitative immunoblotting, as in Figure 4B.

Timothy P. Levine, et al. Mol Biol Cell. 2000 July;11(7):2267-2281.
2.
Figure 3

Figure 3. From: Inositol Phosphorylceramide Synthase Is Located in the Golgi Apparatus of Saccharomyces cerevisiae.

Localization of epitope-tagged Aur1p by immunofluorescence. (A) Immunofluorescence confocal micrographs of yeast cells in which the AUR1 gene is either tagged with HA or not (control) and probed with the rat anti-HA mAb 3F10. (B) Double-label immunofluorescence confocal micrographs comparing the localization of Aur1p-HA with that of endogenous Anp1p (cis Golgi), myc-tagged Mnt1p (medial Golgi), or GFP-tagged Sec7p (TGN). Anti-HA staining was with a rabbit antiserum or, for colocalization with Anp1p, rat mAb 3F10. Most of the Aur1p-positive structures were also positive for Mnt1p, but a few showed colocalization with Anp1p (arrowheads pointing right). Bars, 2 μm.

Timothy P. Levine, et al. Mol Biol Cell. 2000 July;11(7):2267-2281.
3.
Figure 2

Figure 2. From: Inositol Phosphorylceramide Synthase Is Located in the Golgi Apparatus of Saccharomyces cerevisiae.

Epitope-tagged Aur1p cofractionates with the Golgi apparatus. (A) Immunoblot of total proteins from strain SEY6210 either with (HA) or without (wt) three copies of the HA epitope tag inserted at the COOH terminus of the AUR1 gene. The background band is characteristic of the 12CA5 anti-HA mAb. (B) Immunoblot of membranes from a strain expressing Aur1p-HA separated by velocity sedimentation and probed with 12CA5 and endogenous markers for ER, vacuole (vac), and Golgi, followed by peroxidase-conjugated secondary antibodies for visualization by chemiluminescence. Mobilities of size markers are indicated (kDa), and the faint band below BiP is protein disulfide isomerase, which also reacts with the anti-HDEL mAb 2E7.

Timothy P. Levine, et al. Mol Biol Cell. 2000 July;11(7):2267-2281.
4.
Figure 1

Figure 1. From: Inositol Phosphorylceramide Synthase Is Located in the Golgi Apparatus of Saccharomyces cerevisiae.

Hydropathy plots of the bilayer-spanning regions of single TMD proteins from the organelles of the yeast secretory pathway. (A) The known single TMD proteins of the ER and of the cis and medial Golgi (Table 2) were aligned by the beginning of the TMDs on the cytoplasmic side (residue 1 on the plot, defined as being the residue after the last strongly hydrophilic residue of the cytoplasmic tail). For each position in each aligned set, the average side chain hydropathy was calculated with the use of the Goldman, Engelman, Steitz scale (kcal/mol; Engelman et al., 1986). The statistical significance of differences was evaluated with the use of a two-sample t test (Satterthwaite's method to allow for unequal variances). (B) As in A, except that the proteins were from the medial Golgi, TGN, and plasma membrane (PM).

Timothy P. Levine, et al. Mol Biol Cell. 2000 July;11(7):2267-2281.
5.
Figure 6

Figure 6. From: Inositol Phosphorylceramide Synthase Is Located in the Golgi Apparatus of Saccharomyces cerevisiae.

Aur1p-HA does not return to the ER when ER-to-Golgi transport is blocked. (A) Double-label immunofluorescence confocal micrographs of SEY6210 (wild type) and RSY282 (sec23-1) cells expressing both HA-tagged Aur1p and myc-tagged Emp47p and probed with rabbit anti-HA and a mouse anti-myc mAb. Cells were shifted to 37°C for 1 h or left at 25°C before fixation as indicated. (B) Quantitation of the amount of Aur1p-HA (Aur1) and Emp47-Myc in P100 membrane fractions. Cells of the indicated strains were treated as in A, with the addition of cycloheximide (67 μg/ml) from 10 min before temperature shift to prevent the accumulation of newly made proteins in the ER. Membranes were isolated and fractionated as described by Holthuis et al. (1998), and the tagged proteins were quantified as in Figure 4.

Timothy P. Levine, et al. Mol Biol Cell. 2000 July;11(7):2267-2281.
6.
Figure 7

Figure 7. From: Inositol Phosphorylceramide Synthase Is Located in the Golgi Apparatus of Saccharomyces cerevisiae.

Modification of C6-NBD-ceramide by living yeast cells. (A) TLC separation of the products of lipids C6-NBD-ceramide extracted either from microsomes (memb), as in Figure 4, or from live yeast incubated with C6-NBD-ceramide, as described in MATERIALS AND METHODS. AbA treatment was at 10 μg/ml, with a 10-min preincubation for the cells. The faster migrating band is the precursor C6-NBD-ceramide. (B) As in A except that wild-type (SEY6211; wt) and sec6-4 (JWY47; sec6) cells were incubated with C6-NBD-ceramide for 10 min at 25°C and then for 5 min at 37°C. After washing and back-extraction for 60 min on ice, cells were resuspended into fresh, prewarmed medium containing 10 mg/ml defatted BSA and 10 μg/ml AbA and incubated for 10 min at 37°C, and lipids were extracted from cells and medium. (C) Live yeast labeled with C6-NBD-ceramide with or without AbA, as described in MATERIALS AND METHODS, and photographed with both fluorescence and Nomarski optics. Rings can be seen around the nucleus (n) distinct from the vacuole (v). Bar, 1.5 μm.

Timothy P. Levine, et al. Mol Biol Cell. 2000 July;11(7):2267-2281.
7.
Figure 4

Figure 4. From: Inositol Phosphorylceramide Synthase Is Located in the Golgi Apparatus of Saccharomyces cerevisiae.

Modification of C6-NBD-ceramide by Aur1p. (A) TLC separation of the reaction products produced when C6-NBD-ceramide was incubated with gradient fractions containing Golgi membranes, as described in MATERIALS AND METHODS. The samples were diluted threefold serially from the most active membrane fractions in the gradient shown in Figure 5 (81; 25 μg/ml membranes), and the Aur1p inhibitor AbA was added to a sample containing the highest concentration of membranes (10 μg/ml AbA). The upper band corresponds to unmodified C6-NBD-ceramide, and the faint AbA-insensitive band (*) comigrates with C6-NBD fatty acid and is apparently released from C6-NBD-ceramide during the acid extraction of lipids before TLC. (B) IPC synthase activity cofractionates with Golgi and not ER membranes. Fractions from a velocity gradient separation of membranes from SEY6210 were assayed for the indicated proteins by quantitative immunoblotting with the use of 125I-protein A (Amersham, Arlington Heights, IL) and a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Fractions were also incubated with C6-NBD-ceramide, the products were separated by TLC, and the modified band was quantified with the use of a fluorescence imager. The IPC synthase activity is the mean of results from four gradients, and the peak is slightly broadened as a result.

Timothy P. Levine, et al. Mol Biol Cell. 2000 July;11(7):2267-2281.
8.
Figure 8

Figure 8. From: Inositol Phosphorylceramide Synthase Is Located in the Golgi Apparatus of Saccharomyces cerevisiae.

The COOH terminus of Aur1p is cytoplasmic. (A) Sequences of the last three TMDs of Aur1p and its homologues (black bars X, Y, and Z, as predicted by PHDhtm [Rost et al., 1996]). Residues identical (black) or related (gray) in 4 or more of the 11 sequences are shaded. A 124-residue hydrophilic loop found only in Ipt1p (residues 313–436) is omitted (arrowhead). Asterisks indicate the conserved motifs originally found in integral membrane phosphatases and soluble haloperoxidases (Neuwald, 1997), and the circled asterisk indicates histidine 294, which was mutated to alanine. (B) Arrangement of the motifs and the COOH terminus with respect to the last three TMDs for Aur1p and the ZZ-tagged version. (C) Immunoblot of membranes from yeast expressing Aur1p or Van1p with a TEV-cleavable ZZ domain at the COOH terminus. Membranes were treated for 1 h at 15°C with 20 U of TEV protease (Life Technologies, Grand Island, NY) and 0.4% Triton X-100 (Tx100), as indicated. Each lane contains membranes prepared from 40 OD (600 nm) units of log-phase cells, as described previously (Holthuis et al., 1998). Membrane protein Bet1p was examined to demonstrate constant protein recovery. The apparent reduction in Aur1p-ZZ cleavage in the presence of Triton X-100 was seen reproducibly and may reflect partial masking of the cleavage site in detergent micelles.

Timothy P. Levine, et al. Mol Biol Cell. 2000 July;11(7):2267-2281.

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