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. 2017 Mar 28;29(4):890–911. doi: 10.1105/tpc.17.00056

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© 2017 American Society of Plant Biologists. All rights reserved.

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Figure 7. — ATG6 Is a Target of SINAT1, SINAT2, SINAT5, and SINAT6.

(A) Y2H analysis showing the interaction between ATG6 and SINAT proteins. ATG6-AD and SINAT-BD (SINAT1-BD, SINAT2-BD, SINAT3-BD, SINAT4-BD, SINAT5-S1-BD, and SINAT6-BD) were coexpressed in yeast and selected on SD/-Trp-Leu-His-Ade medium (–LWH) containing 5 mM 3-amino-1,2,4-triazole. AD indicates empty AD plasmid.

(B) In vivo CoIP analysis showing the physical interaction between ATG6 and SINATs. HA-tagged ATG6 (ATG6-HA) was coexpressed with FLAG-tagged SINATs (SINAT1-FLAG, SINAT2-FLAG, SINAT3-FLAG, SINAT4-FLAG, SINAT5-S1-FLAG, and SINAT6-FLAG) in Arabidopsis protoplasts and immunoprecipitated by FLAG affinity magnetic beads.

(C) Truncation analysis of SINAT5 to identify the functional domain mediating the ATG6-SINAT5 association. Full-length SINAT5 amplified from ecotype Landsberg erecta containing a RING finger (RING), a zinc finger (ZINC), and a TRAF domain (TRAF). SINAT5-S1 and SINAT5-S2 are two alternatively spliced products of ecotype Col-0 containing a TRAF domain with impaired RING or ZINC domains. △183-309 and △1-159 are artificial truncated proteins without the TRAF domain or RING/ZINC domains. Truncated SINAT5 was fused to the BD domain and coexpressed with ATG6-AD in yeast. Positive clones were selected on SD medium lacking Trp, Leu, His, and Ade (–LWH). AD indicates empty AD plasmid.

(D) In vivo CoIP assay showing the association between ATG6-HA and FLAG-tagged SINAT5 truncations (SINAT5-S1, SINAT5-S2, SINAT5△183-309, and SINAT5△1-159). The plasmids were coexpressed in Arabidopsis protoplasts and immunoprecipitated by FLAG affinity magnetic beads.

(E) In vitro ubiquitination assay of SINAT1 and SINAT2. Recombinant proteins MBP-SINAT1 and MBP-SINAT2 purified from Escherichia coli were assayed for E3 activity in the presence of wheat E1, human E2, and ubiquitin, as indicated.

(F) In vivo ubiquitination of ATG6 by SINAT1, SINAT2, SINAT5-S1, and SINAT6. ATG6-HA was coexpressed with SINAT1-FLAG, SINAT2-FLAG, SINAT5-S1-FLAG, or SINAT6-FLAG in Arabidopsis protoplasts, and its ubiquitination was detected by protein blot analysis. The asterisk indicates an unidentified nonspecific band in the vector control.

(G) Effects of SINAT1, SINAT2, SINAT5-S1, and SINAT6 on ATG6 protein stability. ATG6-HA was coexpressed with various amounts (0, 10, 20, and 30 μg) of SINAT1-FLAG, SINAT2-FLAG, SINAT5-S1-FLAG, or SINAT6-FLAG for 16 h, and the blots were probed with anti-HA and anti-FLAG antibodies. Relative intensity of each protein normalized to the loading control is shown below. The numbers on the left indicate the molecular mass (kD) of each size marker.