Journal:

Article Title: Apg7p/Cvt2p: A Novel Protein-activating Enzyme Essential for Autophagy

doi:

Figure Lengend Snippet: Apg12p conjugates with Apg7p via a thioester bond. (A) Apg12p was coimmunoprecipitated with Apg7p. pAPG7myc-314 plasmid (CEN) was transformed into apg7Δ cells producing HA-tagged Apg12p to express c-myc–tagged Apg7p. A transformant was designated as the YIT702 strain. The pRS314 vector was used as a control. Cell lysates were prepared as described in MATERIALS AND METHODS. A c-myc–tagged Apg7p was immunoprecipitated with Agarose beads conjugated with anti–c-myc mAb (9E10). The immunoprecipitates were subjected to SDS-PAGE on a 10% gel and transferred to a polyvinylidene difluoride membrane. Proteins were detected by Western blotting with αApg7N antibody (for Apg7p) and anti-HA mouse mAb (16B12; for HA-tagged Apg12p). (B) The coimmunoprecipitation of Apg12p with Apg7p is sensitive to DTT. A cell lysate of the YIT702 strain was prepared as described above. The lysate was boiled with (DTT+) or without 1 mM DTT (DTT−). Immunoprecipitation and Western blotting were performed as described above. (C) The higher molecular weight band of Apg7p in cells overexpressing Apg7p and Apg12p is sensitive to β-mercaptoethanol. Cells grown to early logarithmic phase in MVD medium were harvested and converted to spheroplasts in spheroplasting solution. The spheroplasts were harvested in 1.3 M sorbitol as a cushion, lysed with a 4× SDS sample buffer (Ausubel et al., 1995 ) with a protease-inhibitor mixture (Sigma). The lysate was boiled for 5 min in the presence (βME+) or absence (βME−) of 3% β-mercaptoethanol. SDS-PAGE on a 7% gel and Western blotting wereperformed as described above. pYO324/pAPG12HA-426: strain YIT704 cells; pAPG7myc-314/pAPG12HA-316: strain YIT702 cells; pAPG7myc-324/pAPG12HA-426: strain YIT703 cells.

Article Snippet: Oligonucleotides were synthesized by Sawady Technology (Tokyo, Japan) or ESPEC oligo-service (Ibaraki, Japan). pYO324 was a kind gift from Y. Ohya, pRS series vectors were kind gifts from P. Hieter, and pGAD-C1 vector, pGBD-C1 vector, and PJ69-4A strain were kind gifts from P. James ( Sikorski and Hieter, 1989 ; James et al. , 1996 ; Homma et al. 1998 ). pGEX-3X and cyanogen bromide-activated Sepharose beads were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden); pGEM-T was from Promega (Madison, WI). table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Genotype Source Yeast strains YW5-1B MATa leu2-3, 112 trp1 ura3-52 Tsukada and Ohsumi (1993) YIT701 MATa leu2-3, 112 trp1 ura3-52 apg7Δ::LEU2 This study YIT702 MATa leu2-3, 112 trp1 ura3-52 apg7Δ::LEU2 This study [pAPG7myc-314, pAPG12HA-316] YIT703 MATa leu2-3, 112 trp1 ura3-52 apg7Δ::LEU2 This study [pAPG7myc-324, pAPG12HA-426] YIT704 MATa leu2-3, 112 trp1 ura3-52 apg7Δ::LEU2 This study [pYO324, pAPG12HA-426] YIT7G333A MATa leu2-3, 112 trp1 ura3-52 apg7Δ::LEU2 This study [pAPG7G333Amyc-314, pAPG12HA-316] YIT7C507A MATa leu2-3, 112 trp1 ura3-52 apg7Δ::LEU2 This study [pAPG7C507 Amyc-314, pAPG12HA-316] YTS2 MATa ura3-52 lys2-801 ade2-101 trp1 his3 This study leu2 pho8::pho8Δ60 apg7::HIS3 PJ69-4A MATa trp1-901 leu2-3, 112 ura3-52 his3-200 James et al. (1996) gal4Δ gal80Δ GAL2-ADE2 LYS2::GAL1-HIS3 met2::GAL7-lacZ Plasmids pRS314 CEN6 TRP1 Sikorski and Hieter (1989) pRS316 CEN6 URA3 Sikorski and Hieter (1989) pYO324 2μ TRP1 Homma et al. (1998) pAPG7myc-314 CEN6 TRP1 APG7-3 × cmyc epitope This study pAPG7myc-324 2μ TRP1 APG7-3 × cmyc epitope This study pAPG7G333Amyc-314 CEN6 TRP1 APG7 G333A -3 × cmyc epitope This study pAPG7C507Amyc-314 CEN6 TRP1 APG7 C507A -3 × cmyc epitope This study pAPG12HA-316 CEN6 URA3 3 × HA epitope-APG12 Mizushima et al. (1998) pAPG12HA-426 2μ URA3 3 × HA epitope-APG12 Mizushima et al. (1998) pGAD-C1 2μ LEU2 P ADH1 ::GAL4 AD James et al. (1996) pGAD-APG12 2μ LEU2 P ADH1 ::GAL4 AD-APG12 This study pGBD-C1 2μ TRP1 P ADH1 ::GAL4 BD James et al. (1996) pGBD-APG7 2μ TRP1 P ADH1 ::GAL4 BD-APG7-3 × cmyc epitope This study pGBD-APG7G333A 2μ TRP1 P ADH1 ::GAL4 BD-APG7 G333A -3 × cmyc epitope This study pGBD-APG7C507A 2μ TRP1 P ADH1 ::GAL4 BD-APG7 C507A -3 × cmyc epitope This study Open in a separate window Yeast strains and plasmids

Techniques: Plasmid Preparation, Transformation Assay, Immunoprecipitation, SDS Page, Western Blot, Molecular Weight, Protease Inhibitor

Journal:

Article Title: Septins, under Cla4p Regulation, and the Chitin Ring Are Required for Neck Integrity in Budding Yeast

doi: 10.1091/mbc.E02-08-0547

Figure Lengend Snippet: Strains used in this study

Article Snippet: To construct pAS8, the CHS2 ORF contained in the multiple cloning site of YEp352 (pEC2, Ford et al. , 1996 ) was cut out with Eco RV and Sac I and ligated to Sma I/ Sac I digested vector pRS314. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Plasmid Description Source Library in pRS200 ATCC 77164 pΔADE2 p[ ade2::hisG::URA3::hisG ] Aparicio et al. ( 1991 ) (ATCC 99604) pEC2 YEp352 CHS2 Ford et al. ( 1996 ) pEC28 pRS412 CHS3 Schmidt et al. ( 2002 ) pEL45 pBluescript KS+ ste20::URA3 Leberer et al. ( 1992 ) pFD26 cla4::LEU2 Cvrcková et al. ( 1995 ) pFLC1 p GALISTE20 S. Davis pHV7-37 YCp50 chs3R995A C. Roncero pHV8 YEp352 CHS3 Valdivieso et al. ( 1991 ) pLP17 pRS315 CDC12-GFP Lippincott and Li ( 1998b ) pNKY50 YEp24 hisG::URA3::hisG Alani et al. ( 1987 ) pSM491 Contains triple HA tag S. Michaelis pMS17 pRS200 CLA4 This study pMS32 pRS200 cla4::URA3 This study pMS46 pRS200 cla4::TRP1 This study pMS39 From pRS200 library This study pMS55 pRS316 MYO1 -GFP Schmidt et al. ( 2002 ) pMS63 pRS426 MYO1 -GFP This study pMS75 YEp352 chs3R995A This study pMS76 pRS313 CDC12 -GFP This study pMS79 YEp352 chs3R995A::HA This study pMS80 YEp352 CHS3::HA This study pRS316CDC3GFP CDC3 -GFP M. Longtine p366 YCp50 with URA3 replaced by LEU2 ATCC 77163 Library in p366 ATCC 77162 pFD10 YCp50 ADE3 A. Bender pAS8 pRS314 CHS2 This laboratory pLP8 pRS315 MYO1-GFP Lippincott and Li, ( 1998a ) p19 From p366 library This study pAV1 YCp50 CHS3ADE3 This study pAV4 YCp50( URA3 cut out) + ade3::hisG-URA3-hisG This study pAV10 YEp351 leu2::TRP1 This study pAV11 p366 mog1HOC1CDC11mig1 This study pAV12 YEp351 CDC11 This study pAV13 p366 CDC11 This study pAV17 YEp351 cdc11-25 This study pAV21 pRS425 MYO1-GFP This study Open in a separate window Plasmids used in this study To obtain pMS76, the LEU2- containing Pvu I fragment from pLP17 ( Lippincott and Li, 1998b ) was replaced with the HIS3- containing Pvu I fragment from pRS313.

Techniques:

Journal:

Article Title: Septins, under Cla4p Regulation, and the Chitin Ring Are Required for Neck Integrity in Budding Yeast

doi: 10.1091/mbc.E02-08-0547

Figure Lengend Snippet: Plasmids used in this study

Article Snippet: To construct pAS8, the CHS2 ORF contained in the multiple cloning site of YEp352 (pEC2, Ford et al. , 1996 ) was cut out with Eco RV and Sac I and ligated to Sma I/ Sac I digested vector pRS314. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Plasmid Description Source Library in pRS200 ATCC 77164 pΔADE2 p[ ade2::hisG::URA3::hisG ] Aparicio et al. ( 1991 ) (ATCC 99604) pEC2 YEp352 CHS2 Ford et al. ( 1996 ) pEC28 pRS412 CHS3 Schmidt et al. ( 2002 ) pEL45 pBluescript KS+ ste20::URA3 Leberer et al. ( 1992 ) pFD26 cla4::LEU2 Cvrcková et al. ( 1995 ) pFLC1 p GALISTE20 S. Davis pHV7-37 YCp50 chs3R995A C. Roncero pHV8 YEp352 CHS3 Valdivieso et al. ( 1991 ) pLP17 pRS315 CDC12-GFP Lippincott and Li ( 1998b ) pNKY50 YEp24 hisG::URA3::hisG Alani et al. ( 1987 ) pSM491 Contains triple HA tag S. Michaelis pMS17 pRS200 CLA4 This study pMS32 pRS200 cla4::URA3 This study pMS46 pRS200 cla4::TRP1 This study pMS39 From pRS200 library This study pMS55 pRS316 MYO1 -GFP Schmidt et al. ( 2002 ) pMS63 pRS426 MYO1 -GFP This study pMS75 YEp352 chs3R995A This study pMS76 pRS313 CDC12 -GFP This study pMS79 YEp352 chs3R995A::HA This study pMS80 YEp352 CHS3::HA This study pRS316CDC3GFP CDC3 -GFP M. Longtine p366 YCp50 with URA3 replaced by LEU2 ATCC 77163 Library in p366 ATCC 77162 pFD10 YCp50 ADE3 A. Bender pAS8 pRS314 CHS2 This laboratory pLP8 pRS315 MYO1-GFP Lippincott and Li, ( 1998a ) p19 From p366 library This study pAV1 YCp50 CHS3ADE3 This study pAV4 YCp50( URA3 cut out) + ade3::hisG-URA3-hisG This study pAV10 YEp351 leu2::TRP1 This study pAV11 p366 mog1HOC1CDC11mig1 This study pAV12 YEp351 CDC11 This study pAV13 p366 CDC11 This study pAV17 YEp351 cdc11-25 This study pAV21 pRS425 MYO1-GFP This study Open in a separate window Plasmids used in this study To obtain pMS76, the LEU2- containing Pvu I fragment from pLP17 ( Lippincott and Li, 1998b ) was replaced with the HIS3- containing Pvu I fragment from pRS313.

Techniques:

Journal:

Article Title: Septins, under Cla4p Regulation, and the Chitin Ring Are Required for Neck Integrity in Budding Yeast

doi: 10.1091/mbc.E02-08-0547

Figure Lengend Snippet: Morphology of the cdc11-25 mutant isolated in the screen for synthetic lethality with chs3-1 and of related strains. (A and B) AVY2–25 (cdc11-25) at 26°C in minimal and YEPD medium, respectively. (C and D) AVY2–25 at 30°C in minimal and YEPD medium, respectively. (E and F) AVY5 (cdc11-25 chs3-1) at 26°C in minimal and YEPD medium, respectively. (G) AVY2–25 transformed with pAV13 (YEp351CDC11) at 30°C in minimal medium. (H) AVY3 (cdc11-25 cla4:: LEU2) at 26°C in minimal medium.

Article Snippet: To construct pAS8, the CHS2 ORF contained in the multiple cloning site of YEp352 (pEC2, Ford et al. , 1996 ) was cut out with Eco RV and Sac I and ligated to Sma I/ Sac I digested vector pRS314. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Plasmid Description Source Library in pRS200 ATCC 77164 pΔADE2 p[ ade2::hisG::URA3::hisG ] Aparicio et al. ( 1991 ) (ATCC 99604) pEC2 YEp352 CHS2 Ford et al. ( 1996 ) pEC28 pRS412 CHS3 Schmidt et al. ( 2002 ) pEL45 pBluescript KS+ ste20::URA3 Leberer et al. ( 1992 ) pFD26 cla4::LEU2 Cvrcková et al. ( 1995 ) pFLC1 p GALISTE20 S. Davis pHV7-37 YCp50 chs3R995A C. Roncero pHV8 YEp352 CHS3 Valdivieso et al. ( 1991 ) pLP17 pRS315 CDC12-GFP Lippincott and Li ( 1998b ) pNKY50 YEp24 hisG::URA3::hisG Alani et al. ( 1987 ) pSM491 Contains triple HA tag S. Michaelis pMS17 pRS200 CLA4 This study pMS32 pRS200 cla4::URA3 This study pMS46 pRS200 cla4::TRP1 This study pMS39 From pRS200 library This study pMS55 pRS316 MYO1 -GFP Schmidt et al. ( 2002 ) pMS63 pRS426 MYO1 -GFP This study pMS75 YEp352 chs3R995A This study pMS76 pRS313 CDC12 -GFP This study pMS79 YEp352 chs3R995A::HA This study pMS80 YEp352 CHS3::HA This study pRS316CDC3GFP CDC3 -GFP M. Longtine p366 YCp50 with URA3 replaced by LEU2 ATCC 77163 Library in p366 ATCC 77162 pFD10 YCp50 ADE3 A. Bender pAS8 pRS314 CHS2 This laboratory pLP8 pRS315 MYO1-GFP Lippincott and Li, ( 1998a ) p19 From p366 library This study pAV1 YCp50 CHS3ADE3 This study pAV4 YCp50( URA3 cut out) + ade3::hisG-URA3-hisG This study pAV10 YEp351 leu2::TRP1 This study pAV11 p366 mog1HOC1CDC11mig1 This study pAV12 YEp351 CDC11 This study pAV13 p366 CDC11 This study pAV17 YEp351 cdc11-25 This study pAV21 pRS425 MYO1-GFP This study Open in a separate window Plasmids used in this study To obtain pMS76, the LEU2- containing Pvu I fragment from pLP17 ( Lippincott and Li, 1998b ) was replaced with the HIS3- containing Pvu I fragment from pRS313.

Techniques: Mutagenesis, Isolation, Transformation Assay

Journal:

Article Title: Septins, under Cla4p Regulation, and the Chitin Ring Are Required for Neck Integrity in Budding Yeast

doi: 10.1091/mbc.E02-08-0547

Figure Lengend Snippet: Septin localization in different strains

Article Snippet: To construct pAS8, the CHS2 ORF contained in the multiple cloning site of YEp352 (pEC2, Ford et al. , 1996 ) was cut out with Eco RV and Sac I and ligated to Sma I/ Sac I digested vector pRS314. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Plasmid Description Source Library in pRS200 ATCC 77164 pΔADE2 p[ ade2::hisG::URA3::hisG ] Aparicio et al. ( 1991 ) (ATCC 99604) pEC2 YEp352 CHS2 Ford et al. ( 1996 ) pEC28 pRS412 CHS3 Schmidt et al. ( 2002 ) pEL45 pBluescript KS+ ste20::URA3 Leberer et al. ( 1992 ) pFD26 cla4::LEU2 Cvrcková et al. ( 1995 ) pFLC1 p GALISTE20 S. Davis pHV7-37 YCp50 chs3R995A C. Roncero pHV8 YEp352 CHS3 Valdivieso et al. ( 1991 ) pLP17 pRS315 CDC12-GFP Lippincott and Li ( 1998b ) pNKY50 YEp24 hisG::URA3::hisG Alani et al. ( 1987 ) pSM491 Contains triple HA tag S. Michaelis pMS17 pRS200 CLA4 This study pMS32 pRS200 cla4::URA3 This study pMS46 pRS200 cla4::TRP1 This study pMS39 From pRS200 library This study pMS55 pRS316 MYO1 -GFP Schmidt et al. ( 2002 ) pMS63 pRS426 MYO1 -GFP This study pMS75 YEp352 chs3R995A This study pMS76 pRS313 CDC12 -GFP This study pMS79 YEp352 chs3R995A::HA This study pMS80 YEp352 CHS3::HA This study pRS316CDC3GFP CDC3 -GFP M. Longtine p366 YCp50 with URA3 replaced by LEU2 ATCC 77163 Library in p366 ATCC 77162 pFD10 YCp50 ADE3 A. Bender pAS8 pRS314 CHS2 This laboratory pLP8 pRS315 MYO1-GFP Lippincott and Li, ( 1998a ) p19 From p366 library This study pAV1 YCp50 CHS3ADE3 This study pAV4 YCp50( URA3 cut out) + ade3::hisG-URA3-hisG This study pAV10 YEp351 leu2::TRP1 This study pAV11 p366 mog1HOC1CDC11mig1 This study pAV12 YEp351 CDC11 This study pAV13 p366 CDC11 This study pAV17 YEp351 cdc11-25 This study pAV21 pRS425 MYO1-GFP This study Open in a separate window Plasmids used in this study To obtain pMS76, the LEU2- containing Pvu I fragment from pLP17 ( Lippincott and Li, 1998b ) was replaced with the HIS3- containing Pvu I fragment from pRS313.

Techniques:

Journal:

Article Title: Septins, under Cla4p Regulation, and the Chitin Ring Are Required for Neck Integrity in Budding Yeast

doi: 10.1091/mbc.E02-08-0547

Figure Lengend Snippet: Septin defects in cdc11-25. Strains were grown in minimal medium at 30°C. A–D, fluorescence images; E–H, Nomarski images. (A, B, E, and F) Strain AVY2–25[pLP17] (cdc11-25); (C, D, G, and H) strain AVY5[pRS316-CDC3-GFP] (cdc11-25 chs3-1).

Article Snippet: To construct pAS8, the CHS2 ORF contained in the multiple cloning site of YEp352 (pEC2, Ford et al. , 1996 ) was cut out with Eco RV and Sac I and ligated to Sma I/ Sac I digested vector pRS314. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Plasmid Description Source Library in pRS200 ATCC 77164 pΔADE2 p[ ade2::hisG::URA3::hisG ] Aparicio et al. ( 1991 ) (ATCC 99604) pEC2 YEp352 CHS2 Ford et al. ( 1996 ) pEC28 pRS412 CHS3 Schmidt et al. ( 2002 ) pEL45 pBluescript KS+ ste20::URA3 Leberer et al. ( 1992 ) pFD26 cla4::LEU2 Cvrcková et al. ( 1995 ) pFLC1 p GALISTE20 S. Davis pHV7-37 YCp50 chs3R995A C. Roncero pHV8 YEp352 CHS3 Valdivieso et al. ( 1991 ) pLP17 pRS315 CDC12-GFP Lippincott and Li ( 1998b ) pNKY50 YEp24 hisG::URA3::hisG Alani et al. ( 1987 ) pSM491 Contains triple HA tag S. Michaelis pMS17 pRS200 CLA4 This study pMS32 pRS200 cla4::URA3 This study pMS46 pRS200 cla4::TRP1 This study pMS39 From pRS200 library This study pMS55 pRS316 MYO1 -GFP Schmidt et al. ( 2002 ) pMS63 pRS426 MYO1 -GFP This study pMS75 YEp352 chs3R995A This study pMS76 pRS313 CDC12 -GFP This study pMS79 YEp352 chs3R995A::HA This study pMS80 YEp352 CHS3::HA This study pRS316CDC3GFP CDC3 -GFP M. Longtine p366 YCp50 with URA3 replaced by LEU2 ATCC 77163 Library in p366 ATCC 77162 pFD10 YCp50 ADE3 A. Bender pAS8 pRS314 CHS2 This laboratory pLP8 pRS315 MYO1-GFP Lippincott and Li, ( 1998a ) p19 From p366 library This study pAV1 YCp50 CHS3ADE3 This study pAV4 YCp50( URA3 cut out) + ade3::hisG-URA3-hisG This study pAV10 YEp351 leu2::TRP1 This study pAV11 p366 mog1HOC1CDC11mig1 This study pAV12 YEp351 CDC11 This study pAV13 p366 CDC11 This study pAV17 YEp351 cdc11-25 This study pAV21 pRS425 MYO1-GFP This study Open in a separate window Plasmids used in this study To obtain pMS76, the LEU2- containing Pvu I fragment from pLP17 ( Lippincott and Li, 1998b ) was replaced with the HIS3- containing Pvu I fragment from pRS313.

Techniques: Fluorescence