Arabidopsis PIP2 isoforms prevent the post-translational repression of PIP1 aquaporins

Jhala, Komal (2024): Arabidopsis PIP2 isoforms prevent the post-translational repression of PIP1 aquaporins. Dissertation, LMU München: Faculty of Chemistry and Pharmacy

Abstract

Plant plasma membrane aquaporins (plasma membrane intrinsic proteins, PIPs) facilitate the exchange of water and other non-charged molecules across the cell membrane. PIPs are divided into PIP1 and PIP2 subclasses. Arabidopsis thaliana encodes five PIP1 and eight PIP2 isoforms. They are synthesized at the endoplasmic reticulum (ER), aggregate into tetramers, and are routed to the plasma membrane (PM). PIP1 and PIP2 are co-regulated at various levels, which may coordinate and/or adjust their function(s), e.g., certain PIP2s have been shown to assist specific PIP1s to reach the plasmalemma. In addition, there were indications that the loss of PIP2 isoforms downregulated PIP1 proteins instead of a compensating response. pip2 multiple mutants were employed to understand this phenomenon and the contribution of specific PIP2 isoforms towards the expression of PIP1 proteins. PIP2;1, PIP2;2, and PIP2;7 turned out to be the major isoforms stabilizing PIP1s. PIP1 protein level was reduced by about 80% in the triple mutant pip2;1 pip2;2 pip2;7 compared to wild-type plants. This repression was established post-translationally, since PIP1’s steady-state transcripts were not affected and PIP1 mRNAs were associated in a similar manner with protein-synthesizing polysomes in the triple mutant as wild type. Thus, the degradative pathway(s) involved in this post-translational processing of PIP1 proteins were investigated. The Ubiquitin Proteasome System (UPS) and autophagy/vacuole linked degradation are the two major pathways operating in eukaryotes for removal of aberrant proteins. Introgression of HRD1A, HRD1B and DERLIN1 i.e., loss-of-function mutations of UPS-dependent ER Associated Degradation pathway (ERAD) components, into pip2;1 pip2;2 and pip2;1 pip2;2 pip2;7 mutants partially stabilized PIP1 protein level of the pip2;1 pip2;2 pip2;7 mutant. Moreover, the ATG7 mutation known to block autophagy mediated degradation was introduced into pip2;1 pip2;2 and pip2;1 pip2;2 pip2;7 mutants. There was no PIP1 protein stabilization observed in these mutants. Further, pharmacological inhibition of PIP1 protein repression in pip2;1 pip2;2 pip2;7 mutant with MG132 (proteasome inhibitor) and concanamycin A (autophagy inhibitor) yielded evidence for a parallel operation of UPS-dependent ERAD and autophagy/vacuole linked degradation contributing in the post-translational repression of PIP1, which is induced by the lack of PIP2s, potentially due to missing PIP2-PIP1 interactions and/or the failure of PIP1s to efficiently reach the PM. By coupling the regulation of protein abundance, these findings may propose a novel, regulatory mechanism between PIP1s and PIP2s in response to different environmental scenarios or at a particular stage in the plant growth.

• Item Type: Theses (Dissertation, LMU Munich)
• Subjects: 500 Natural sciences and mathematics; 500 Natural sciences and mathematics > 540 Chemistry and allied sciences
• Faculties: Faculty of Chemistry and Pharmacy
• Language: English
• Date of oral examination: 13. August 2024
• 1. Referee: Schäffner, Anton
• Signature of the printed copy: 0001/UMC 32259