Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2021 Feb 26;11:4788. doi: 10.1038/s41598-021-83525-1

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Yeast TORC1 activation via the plant H⁺-ATPase PMA2 requires association of its carboxy-terminal tail with 14-3-3 proteins. (A) GAL1p-PMA1 pma2Δ cells transformed with plasmids expressing (Sc)PMA1, (Np)PMA2^E14D, (Np)PMA2^E14D-T955A, (Np)PMA2^E14D-S938A, (Np)PMA2^E14D-S938D, (Np)PMA2^E14D-T931A, (Np)PMA2^E14D-T931D, or no H⁺-ATPase (-) were grown for three days on solid medium with NH₄⁺ as nitrogen source and Gal or Gluc as carbon source. (B) The same cells as in A, additionally expressing HA-NPR1 from a plasmid, were grown on Gluc NH₄⁺ medium in a microplate reader for 28 h. Data points represent averages of the OD at 660 nm of two biological replicates; error bars represent SD. (C) GAL1p-PMA1 pma2Δ cells expressing, from a plasmid, either (Np)PMA2^E14D, (Np)PMA2^E14D-T955A, (Np)PMA2^E14D-T931A, (Np)PMA2^E14D-T931D, (Np)PMA2^E14D-S938D, (Np)PMA2^E14D-S938A, or (Np)PMA2^{E14D-Δ(887-956)} were spotted in two-fold serial dilutions on solid rich (YPD) or Gluc NH₄⁺ (pH 6.1 or 4) medium and incubated for three days. Equivalent results were obtained when the strains additionally expressed HA-Npr1. (D) GAL1p-PMA1 pma2Δ cells expressing, from plasmids, 6xHis-tagged (Sc)Pma1, (Np)PMA2^E14D, (Np)PMA2^{E14D-Δ(887-956)}, (Np)PMA2^E14D-T955A, (Np)PMA2^E14D-S938A, (Np)PMA2^E14D-S938D, (Np)PMA2^E14D-T931A, or (Np)PMA2^E14D-T931D along with HA-NPR1 were grown on Gluc NH₄⁺. After a shift to Gluc proline medium for four hours, the cells were collected and lysed, and His-tagged proteins were pulled down as described in Materials and Methods. Lysates and pulled-down fractions were immunoblotted with anti-14-3-3 or anti-polyhistidine antibodies. (E) Left. GAL1p-PMA1 pma2Δ cells expressing, from plasmids, either (Np)PMA2^E14D or (Np)PMA2^E14D-T955A along with pHluorin were grown on Gluc NH₄⁺ medium. After a shift to Gluc proline for four hours, the cytosolic pH was monitored with (open symbols) or without (filled symbols) addition of FCCP (20 µM), starting at 1 min (indicated by an arrow on the graph). Average values of three biological replicates are shown, and error bars correspond to SD. Right. Strains and growth conditions as in the left panel, except that the cells expressed HA-NPR1 instead of pHluorin. Culture samples were collected before and 4, 10, and 30 min after addition of FCCP (20 µM) or 30 min after addition of NH₄⁺ (5 mM). Crude extracts were prepared and immunoblotted with anti-HA and anti-Pgk antibodies. (F,G) Experiments similar to those in E, except that other (Np)PMA2^E14D mutants were analyzed, as indicated. Original blots of figure panels D, E, F and G are presented in Fig. S3.