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Results: 7

1.
FIGURE 2.

FIGURE 2. From: Gly-46 and His-50 of Yeast Maltose Transporter Mal21p Are Essential for Its Resistance against Glucose-induced Degradation.

Comparison of amino acid sequences in Mal21p and Mal61p. The Mal61p amino acid sequence is from GenBankTM accession number X17391. The underlined regions are predicted transmembrane domains (46). Amino acid residues of Mal21p are shown where the corresponding residues of Mal61p are inconsistent. The six amino acids that are boxed are inconsistent between Mal31p and Mal21p. Basic amino acids are marked with a plus sign and acidic amino acids with a minus sign.

Haruyo Hatanaka, et al. J Biol Chem. 2009 June 5;284(23):15448-15457.
2.
FIGURE 3.

FIGURE 3. From: Gly-46 and His-50 of Yeast Maltose Transporter Mal21p Are Essential for Its Resistance against Glucose-induced Degradation.

Protein expression levels of Mal21p and Mal61p in cells grown in YPM. Yeast cells of HH206 (expressing Mal21p), HH108 (Mal61p), and HH208 (No transporter) were grown on YPM. Whole cell extracts were prepared from the cells in the logarithmic phase and were separated by SDS-PAGE. Maltose transporter proteins were analyzed by Western blot analysis with anti-Mal61p antibody.

Haruyo Hatanaka, et al. J Biol Chem. 2009 June 5;284(23):15448-15457.
3.
FIGURE 1.

FIGURE 1. From: Gly-46 and His-50 of Yeast Maltose Transporter Mal21p Are Essential for Its Resistance against Glucose-induced Degradation.

Yeast strains carrying MAL21 grows on medium containing 2-DOG. Control strains JH1032, ATCC96955 (carrying MAL61), ATCC20598 (MAL21), and CB11 (MAL31, MAL61, and AGT1) were grown in YPD. Cells were harvested, washed with water, and resuspended in water at OD660 = 2.0. The suspensions and their 10-fold dilutions were spotted onto synthetic complete medium with or without 3 mg liter−1 antimycin A supplemented with 2-DOG (at 0, 0.5, and 1.0 mm). The plates were incubated at 30 °C for 3 days before being photographed.

Haruyo Hatanaka, et al. J Biol Chem. 2009 June 5;284(23):15448-15457.
4.
FIGURE 7.

FIGURE 7. From: Gly-46 and His-50 of Yeast Maltose Transporter Mal21p Are Essential for Its Resistance against Glucose-induced Degradation.

Localization of maltose transporters after exposure of the cells to glucose. Strains HH108 (carrying MAL61), HH207 (MAL61[D46G,L50H]), and HH206 (MAL21) were grown in YPM for 2.5 h at 30 °C. Two fractions of cells corresponding to 10 OD units were collected by centrifugation. One was immediately fixed with formaldehyde. The other was resuspended in YNBDS medium and incubated at 30 °C for 1 h prior to fixation. Immunofluorescence directed to maltose transporters was performed using rabbit anti-Mal61p antibody as the first antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG as the second antibody. The cell margins are indicated by arrowheads.

Haruyo Hatanaka, et al. J Biol Chem. 2009 June 5;284(23):15448-15457.
5.
FIGURE 4.

FIGURE 4. From: Gly-46 and His-50 of Yeast Maltose Transporter Mal21p Are Essential for Its Resistance against Glucose-induced Degradation.

Resistance to glucose-induced degradation of yeast maltose transporters. A, strains HH206 (carrying MAL21), HH108 (MAL61), HH227 (MAL31), and HH208 (Vector) were grown in YPD. Cells were collected, washed with water, and resuspended in water at OD660 = 2.0. Serial 10-fold dilutions of the suspension were spotted on YNBMH containing 2-DOG (at 0.5, 1.0, and 2.0 mm). The plates were incubated at 30 °C for 2 days before being photographed. B, yeast cells HH206 (expressing Mal21p) and HH108 (Mal61p) were pregrown in YPD overnight at 30 °C. They were transferred to and grown in YPM for 2.5 h at 30 °C and were then collected by centrifugation and resuspended in YNBDS medium. The cell suspension was incubated at 30 °C, and 10 OD units of the cells were withdrawn at the times indicated. Whole cell extracts were prepared and analyzed by Western blot analysis using anti-Mal61p antibody.

Haruyo Hatanaka, et al. J Biol Chem. 2009 June 5;284(23):15448-15457.
6.
FIGURE 5.

FIGURE 5. From: Gly-46 and His-50 of Yeast Maltose Transporter Mal21p Are Essential for Its Resistance against Glucose-induced Degradation.

Simultaneous substitutions of Asp-46 → Gly and Leu-50 → His were necessary for resistance to glucose-induced degradation of Mal21p. A, yeast cells HH208 carrying vector DNA, HH108 (carrying MAL61), HH210 (MAL61-D46G), HH209 (MAL61-L50H), HH207 (MAL61-D46G, L50H), and HH206 (MAL21) were grown in YPD. Cells were collected, washed with water, and resuspended in water at OD660 = 2.0. Serial 10-fold dilutions of the suspension were spotted onto YNBMH plate containing 2-DOG (at 0, 0.5, 1.0, and 2.0 mm). The plates were incubated at 30 °C for 2 days before being photographed. B, the cells HH206 (expressing Mal21p), HH207 (Mal61p[Gly-46,His-50]), HH210 (Mal61p[Gly-46]), and HH209 (Mal61p[His-50]) pregrown in YPD overnight at 30 °C were transferred to and grown in YPM for 2.5 h at 30 °C. Cells were harvested and resuspended in YNBDS medium for incubation at 30 °C. The cells of 10 OD units were withdrawn at the times indicated. Whole cell extracts were prepared from the samples and analyzed by Western blot analysis using anti-Mal61p antibody.

Haruyo Hatanaka, et al. J Biol Chem. 2009 June 5;284(23):15448-15457.
7.
FIGURE 6.

FIGURE 6. From: Gly-46 and His-50 of Yeast Maltose Transporter Mal21p Are Essential for Its Resistance against Glucose-induced Degradation.

Modification of Mal61p and Mal21p observed in endocytosis and ubiquitination mutant strains. A, yeast strains 23346c (NPI1) and 27038a (npi1) carrying pJHMAL21 or pJHMAL61 were grown in YPM for 2.5 h at 30 °C. Two fractions of cells corresponding to 10 OD units were collected by centrifugation. One was immediately frozen in ethanol-dry ice. The other was resuspended in YNBDS medium and incubated at 30 °C for 1 h. Strains RH1602 (END4) and RH1597 (end4) carrying pJHMAL21 or pJHMAL61 were treated in the same way, except that they were incubated at a restrictive 37 °C for 30 min prior to incubation in YNBDS medium. Cells were centrifuged and immediately frozen in ethanol-dry ice. Whole cell extracts were prepared and separated with SDS-8% PAGE and analyzed by Western blot analysis using anti-Mal61p antibody. Arrows indicate modified transporter proteins with slower mobilities. B, strain RH1597 (end4) cells carrying pJHMAL61-2HA were incubated in YPM at 37 °C for 30 min and then incubated in YNBDS medium at 30 °C for 1 h. Cells were collected and immediately frozen in ethanol-dry ice. Whole cell extracts were prepared, and immunoprecipitation was conducted with anti-Mal61p antibody. Extracts from the immunoprecipitate were separated with SDS-8% PAGE and analyzed by Western blot analyses using anti-HA antibody or anti-ubiquitin (Ub) antibody. Arrows indicate modified transporter proteins with ubiquitin molecules. The arrowhead indicates a possible phosphorylated form of the transporter protein.

Haruyo Hatanaka, et al. J Biol Chem. 2009 June 5;284(23):15448-15457.

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