The Plant Endoplasmic Reticulum

Persson, Staffan; Harper, Jeff

doi:10.1007/11309680

Cited by 5 publications

(1 citation statement)

References 527 publications

(878 reference statements)

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“…On the other side, the antibody against the vacuolar protease asparginyl endopeptidase labelled the Golgi and the vacuoles, but not the KDELvesicles (KVs). This indicates that KDEL vesicles bypass the Golgi and fuse with PSV (Okamoto, 2006).…”

Section: A Golgi Independent Pathwaymentioning

confidence: 93%

Functions of RMR proteins in intracellular trafficking in the moss "Physcomitrium patens" Mitten (previously "Physcomitrella patens")

Coppola¹

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In this study, I focused on a new family of receptors, called RMRs (Receptor-like Membrane RING-H2) and I tried to investigate their role in the moss Physcomitrium patens Mitten (previously Physcomitrella patens). There is some evidence that in Angiosperms, RMRs are vacuolar receptors for the neutral/storage vacuole that is a compartment where storage proteins and metabolites are accumulated during seeds development or in somatic tissues. It is distinguished from lytic vacuole which has the same functions as animal lysosomes. The five PpRMR genes have been knocked-out, yielding viable material without visible phenotype (Ayachi, 2012). A trafficking phenotype was described by Fahr (2017) who generated the construct Citrine-Cardosin (Ci-Card) composed of the fluorescent protein Citrine fused to the C-terminal vacuolar sorting determinant (ctVSD) from cardosin A (cardosin is addressed to the vacuole in higher plants —Pereira et al., 2013). The fusion protein was delivered to the central vacuole of PpWT but mistargeted in PpRMR-KO lines, indicating that the targeting of this protein to the vacuole depends on PpRMRs. The introduction of this thesis presents the plant endomembrane system, with particular attention to vacuolar transport and ubiquitylation. In the second chapter, I show the techniques used to attempt to detect PpRMRs by Western Blot: our failure may be due to a rapid degradation of these proteins, which could prevent their detection. In the third chapter, I focused on PpRMR2 involvement in ubiquitylation. We hypothesize that PpRMRs are E3 ligases because they are members of the PA-TM-RING protein family. Most of these proteins have an E3 ubiquitin ligase activity in animals (Seroogy et al., 2004; Borchers et al., 2002), for this reason, we think that plant PpRMRs could have this function as well, which could contribute to vacuolar targeting. Indeed, I could confirm that PpRMR2 has an E3 ubiquitin ligase activity. PpRMRs substrates are still unknown in moss thus we have analysed putative candidates supposing that they could be ubiquitylated by PpRMRs. We have tested this hypothesis through in vitro ubiquitylation assays, obtaining ambiguous results. In the fourth chapter, I show preliminary results about the visible phenotype of PpRMR-KO mutants: PpWT and PpRMR-KO lines displayed phenotypic differences in leafy gametophores, which were accentuated upon salt stress exposure. Lastly, I transformed the transgenic lines PpWT/Ci-Card and Pp5KO/Ci-Card with mutated versions of PpRMR2 and analysed their effect on vacuolar transport by confocal microscopy. For most of the constructions tested, the trafficking was perturbed in both lines. Only PpWT/Ci-Card expressing PpRMR2ΔSer (lacking the Serine-Rich motif) displayed a typical vacuolar pattern.

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Section: A Golgi Independent Pathwaymentioning

confidence: 93%

Functions of RMR proteins in intracellular trafficking in the moss "Physcomitrium patens" Mitten (previously "Physcomitrella patens")

Coppola¹

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Arabidopsis peroxin 16 trafficks through the ER and an intermediate compartment to pre-existing peroxisomes via overlapping molecular targeting signals

Karnik

Trelease

2007

Journal of Experimental Botany

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Previously it has been shown that the endogenous Arabidopsis peroxin, AtPEX16, coexisted at steady state in membranes of the endoplasmic reticulum (ER) and peroxisomes. Herein, an ER-to-peroxisome trafficking pathway and the requisite molecular targeting signals for mycAtPEX16 transiently expressed in Arabidopsis and tobacco BY-2 suspension cells are described. Immunofluorescent mycAtPEX16 was observed initially in the cytosol (<2 h) and subsequently (2-4 h) in perinuclear/reticular ER and non-Golgi/non-peroxisome structures termed the ER-peroxisome intermediate compartment. After 4 h, all catalase- and ascorbate peroxidase-containing peroxisomes also possessed mycAtPEX16, indicative of mycAtPEX16 sorting to pre-existing peroxisomes. Incubations of bombarded cells at 15 degrees C, or in brefeldin A at 25 degrees C, resulted in accumulations of mycAtPEX16 within the ER. Following re-equilibration of cold-treated cells at 25 degrees C, or removal of brefeldin A, mycAtPEX16 was observed mainly in the ER-peroxisome intermediate compartment, and later within all of the peroxisomes in both species. Two internal membrane helices and the intervening sequence including the amino acid residues -VRS- were found necessary and sufficient for targeting AtPEX16 first to the ER and then to peroxisomes. Individual targeting signals for these organelles were indistinguishable, indicative of overlapping signal(s). In summary, the trafficking study of AtPEX16 revealed a dynamic link between the ER and pre-existing peroxisomes, which provided novel data in support of an upgraded semi-autonomous peroxisome model portraying participation of the ER in the sorting of certain peroxisome membrane proteins, such as AtPEX16, through an intermediate compartment to pre-existing plant peroxisomes.

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Functional Characterization of Arabidopsis Calreticulin1a: A Key Alleviator of Endoplasmic Reticulum Stress

Christensen

Svensson

Persson

et al. 2008

Plant and Cell Physiology

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The chaperone calreticulin plays important roles in a variety of processes in the endoplasmic reticulum (ER) of animal cells, such as Ca2+ signaling and protein folding. Although the functions of calreticulin are well characterized in animals, only indirect evidence is available for plants. To increase our understanding of plant calreticulins we introduced one of the Arabidopsis isoforms, AtCRT1a, into calreticulin-deficient (crt-/-) mouse embryonic fibroblasts. As a result of calreticulin deficiency, the mouse crt-/- fibroblasts have decreased levels of Ca2+ in the ER and impaired protein folding abilities. Expression of the AtCRT1a in mouse crt-/- fibroblasts rescued these phenotypes, i.e. AtCRT1a restored the Ca2+-holding capacity and chaperone functions in the ER of the mouse crt-/- fibroblasts, demonstrating that the animal sorting machinery was also functional for a plant protein, and that basic calreticulin functions are conserved across the Kingdoms. Expression analyses using a beta-glucuronidase (GUS)-AtCRT1a promoter construct revealed high expression of CRT1a in root tips, floral tissues and in association with vascular bundles. To assess the impact of AtCRT1a in planta, we generated Atcrt1a mutant plants. The Atcrt1a mutants exhibited increased sensitivity to the drug tunicamycin, an inducer of the unfolded protein response. We therefore conclude that AtCRT1a is an alleviator of the tunicamycin-induced unfolded protein response, and propose that the use of the mouse crt-/- fibroblasts as a calreticulin expression system may prove useful to assess functionalities of calreticulins from different species.

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The Plant Endoplasmic Reticulum

Cited by 5 publications

References 527 publications

Functions of RMR proteins in intracellular trafficking in the moss "Physcomitrium patens" Mitten (previously "Physcomitrella patens")

Functions of RMR proteins in intracellular trafficking in the moss "Physcomitrium patens" Mitten (previously "Physcomitrella patens")

Arabidopsis peroxin 16 trafficks through the ER and an intermediate compartment to pre-existing peroxisomes via overlapping molecular targeting signals

Functional Characterization of Arabidopsis Calreticulin1a: A Key Alleviator of Endoplasmic Reticulum Stress

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