Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jul;65(12):3015-27.
doi: 10.1093/jxb/eru147. Epub 2014 Apr 10.

Gamma-secretase subunits associate in intracellular membrane compartments in Arabidopsis thaliana

Michalina Smolarkiewicz  1 , Tomasz Skrzypczak  2 , Michał Michalak  2 , Krzysztof Leśniewicz  2 , J Ross Walker  3 , Gwyneth Ingram  4 , Przemysław Wojtaszek  1
Affiliations

Gamma-secretase subunits associate in intracellular membrane compartments in Arabidopsis thaliana

Michalina Smolarkiewicz et al. J Exp Bot. 2014 Jul.

Abstract

Gamma-secretase is a multisubunit complex with intramembrane proteolytic activity. In humans it was identified in genetic screens of patients suffering from familial forms of Alzheimer's disease, and since then it was shown to mediate cleavage of more than 80 substrates, including amyloid precursor protein or Notch receptor. Moreover, in animals, γ-secretase was shown to be involved in regulation of a wide range of cellular events, including cell signalling, regulation of endocytosis of membrane proteins, their trafficking, and degradation. Here we show that genes coding for γ-secretase homologues are present in plant genomes. Also, amino acid motifs crucial for γ-secretase activity are conserved in plants. Moreover, all γ-secretase subunits: PS1/PS2, APH-1, PEN-2, and NCT colocalize and interact with each other in Arabidopsis thaliana protoplasts. The intracellular localization of γ-secretase subunits in Arabidopsis protoplasts revealed a distribution in endomembrane system compartments that is consistent with data from animal studies. Together, our data may be considered as a starting point for analysis of γ-secretase in plants.

Keywords: Authophagy; complex assembly; endomembrane system; gamma-secretase; intramembrane protease; localization; presenilin; senescence; vesicular trafficking..

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Expression and sequence analysis of γ-secretase subunits. (A) Transcripts for γ-secretase subunits could be detected in different organs of Arabidopsis thaliana. Reverse transcription was conducted with primers specific to each subunit. mRNA was isolated from diverse organs: seedlings, leaves, and stems. Actin 2 was used as internal standard. (B–E) Sequence alignments of amino acid motifs known from animal studies as crucial for γ-secretase activity and complex assembly. (B) APH-1: GXXXG; (C) Nicastrin: XYXGS; (D) PEN-2: WXXNXXF; (E) Presenilins: NF, YD, GXGD, and PALP motifs; (F) Cladogram created on the basis of amino acid sequence alignment for presenilins.
Fig. 2.
Fig. 2.
Cellular localization of γ-secretase subunits. Genetic constructs coding for γ-secretase subunits fused with eGFP were introduced to Arabidopsis thaliana leaf mesophyll protoplasts for transient expression and analysed with confocal microscope. (A) eGFP fluorescence alone was used as control; (C) AtAPH-1–GFP signal is visible in reticular structures; (E) AtPS1–GFP and (G) AtPS2–GFP signal marks vesicular compartments and reticular structures; (I) AtNCT–GFP and (K) AtPEN-2–GFP fluorescence visible mostly in vesicles. Transmitted light images of respective cells are presented next to fluorescent sections (B, D, F, H, J, L). White arrows point to exemplary vesicles. Bar, 10 µm.
Fig. 3.
Fig. 3.
Localization of γ-secretase subunits with markers of intracellular membranous system. AtAPH-1 (A), presenilin 1 (B, C, D), nicastrin (E, F) and AtPEN-2 (G, H) were tagged with eGFP and introduced into Arabidopsis protoplasts simultaneously with marker proteins for different compartments: ER, NIP1;1; trans Golgi network (TGN), VTI12; and prevacuolar compartment (PVC), ARA7 fused with mCherry. Images for fusion with each fluorophore were captured separately and presented in different colours: eGFP in green, mCherry in red. Combined images showing colocalization of two proteins are in yellow. Magnification of areas showing pronounced colocalization is included. AtAPH-1–GFP is localized to the endoplasmic reticulum (A). AtPS1–GFP signal could be detected in ER (B) and TGN (C). However in some cells visibly enlarged compartments showing colocalization of AtPS1–GFP and VTI12–mCherry could be seen (D). AtNCT–GFP is localized to trans Golgi network (E) and prevacuolar compartment (F). Also PEN-2–GFP fluorescence could be detected in both: TGN (G) and PVC (H). White arrows point to exemplary colocalization areas. Bar, 10 µm.
Fig. 4.
Fig. 4.
Fluorescence lifetime measurements of native donor as a control for FLIM-FRET. (A) Fluorescent signal of donor eGFP overexpressed in Arabidopsis thaliana protoplasts, and (B) fluorescence lifetime measurement of eGFP. Mean value of eGFP lifetime is Tamp: 2.45 ns. (C) Fluorescent signal of eGFP fused with AtPEN-2 overexpressed in Arabidopsis protoplasts and (D) fluorescence lifetime measurement of AtPEN-2–GFP. Mean value of AtPEN-2–GFP lifetime is Tamp: 2.36 ns. Fluorescent signal of donor eGFP (E), in the presence of potential acceptor mRFP (F). Merged E and F presented on (G). (H) Donor fluorescence lifetime measurement. Mean value of eGFP lifetime is Tamp: 2.31 ns. (B, D, H) χ2 near 1 indicates good quality of fitting. Bar, 10 µm.
Fig. 5.
Fig. 5.
Interaction studies of γ-secretase subunits in Arabidopsis thaliana protoplasts. Simultaneous expression of γ-secretase subunits showed their colocalization. Fluorescent signal of particular subunit fused with GFP is presented in green and fused with RFP in red. Colocalization of γ-secretase subunits is shown in merged channel (yellow). Fluorescence lifetime measurements are presented on most-right panel and certain lifetime values are presented in white boxes (Tamp). χ2 near 1 indicates good quality of fitting. Bar, 10 µm.
Fig. 6.
Fig. 6.
AtPEN-2N74L shows reticular localization and does not interact with presenilin 1 in Arabidopsis thaliana protoplasts. (A) AtPEN-2N74L–GFP localized to reticular compartment (left) and native form of AtPEN-2–GFP localized to vesicular compartment (miniature, right). Colocalization studies with ER marker protein showed that indeed AtPEN-2N74L–GFP is present in endoplasmic reticulum (B). Moreover, AtPEN-2N74L–GFP fluorescence could no longer be detected in TGN (C) where native form of AtPEN-2 is localized (see Fig. 4G). When co-expressed with mutated AtPEN-2N74L, AtPS2–RFP signal was restricted to reticular compartment (D) and no decrease in fluorescence lifetime could be observed. Bar, 10 µm.
Fig. 7.
Fig. 7.
Presenilin double mutant plants show accelerated chlorosis upon darkness treatment. Leaves of plants from double mutant line ps1/ps2 showed yellowing earlier compared with wild-type plants. This effect could be observed in 6-week-old plants (A), detached leaves (B), and seedlings (C).
Fig. 8.
Fig. 8.
AtPEN-2 is partially localized to autophagosome vesicles in Arabidopsis protoplasts. (A) AtPEN-2 colocalizes with AtATG8, protein marker for autophagosome in plants (white arrow). However, a set of vesicles with AtPEN-2–GFP signal alone could be seen (green arrow) indicating existence of two populations of AtPEN-2 protein. (B) AtPEN-2 did not show colocalization when AtATG8 was not recruited to autophagosome membranes and signal was present in cytoplasm. Bar, 10 µm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Annaert WG, Levesque L, Craessaerts K, Dierinck I, Snellings G, Westaway D, George-Hyslop PS, Cordell B, Fraser P, De Strooper B. 1999. Presenilin 1 controls gamma-secretase processing of amyloid precursor protein in pre-golgi compartments of hippocampal neurons. The Journal of Cell Biology 147, 277–294 - PMC - PubMed
    1. Chakrabarty R, Banerjee R, Chung SM, Farman M, Citovsky V, Hogenhout SA, Tzfira T, Goodin M. 2007. PSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: probing Nicotiana benthamiana-virus interactions. Molecular Plant-Microbe Interactions 20, 740–750 - PubMed
    1. Coen K, Annaert W. 2010. Presenilins: how much more than γ-secretase?! Biochemical Society Transactions 38, 1474–1478 - PubMed
    1. Dries DR, Yu G. 2008. Assembly, maturation, and trafficking of the γ-secretase complex in Alzheimer’s disease. Current Alzheimer Research 5, 132–146 - PMC - PubMed
    1. Fassler M, Li X, Kaether C. 2011. Polar transmembrane-based amino acids in presenilin 1 are involved in endoplasmic reticulum localization, Pen2 protein binding, and γ-secretase complex stabilization. The Journal of Biological Chemistry 286, 38390–38396 - PMC - PubMed

Publication types

MeSH terms

Substances