The major protein import receptor of plastids is essential for chloroplast biogenesis

Two homologous GTP-binding proteins, atToc33 and atToc159, control access of cytosolic precursor proteins to the chloroplast. atToc33 is a constitutive outer chloroplast membrane protein, whereas the precursor receptor atToc159 also exists in a soluble, cytosolic form. This suggests that atToc159 may be able to switch between a soluble and an integral membrane form. By transient expression of GFP fusion proteins, mutant analysis, and biochemical experimentation, we demonstrate that the GTP-binding domain regulates the targeting of cytosolic atToc159 to the chloroplast and mediates the switch between cytosolic and integral membrane forms. Mutant atToc159, unable to bind GTP, does not reinstate a green phenotype in an albino mutant (ppi2) lacking endogenous atToc159, remaining trapped in the cytosol. Thus, the function of atToc159 in chloroplast biogenesis is dependent on an intrinsic GTP-regulated switch that controls localization of the receptor to the chloroplast envelope.

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Targeting of an abundant cytosolic form of the protein import receptor at Toc159 to the outer chloroplast membrane

The Journal of Cell Biology ◽

10.1083/jcb.200104022 ◽

2001 ◽

Vol 154 (2) ◽

pp. 309-316 ◽

Cited By ~ 80

Author(s):

Andreas Hiltbrunner ◽

Jörg Bauer ◽

Pierre-Alexandre Vidi ◽

Sibylle Infanger ◽

Petra Weibel ◽

...

Keyword(s):

Outer Membrane ◽

Large Scale ◽

Protein Import ◽

Protein Translocation ◽

Soluble Form ◽

Chloroplast Biogenesis ◽

Outer Envelope ◽

Precursor Proteins ◽

Outer Envelope Membrane ◽

Import Receptor

Chloroplast biogenesis requires the large-scale import of cytosolically synthesized precursor proteins. A trimeric translocon (Toc complex) containing two homologous GTP-binding proteins (atToc33 and atToc159) and a channel protein (atToc75) facilitates protein translocation across the outer envelope membrane. The mechanisms governing function and assembly of the Toc complex are not yet understood. This study demonstrates that atToc159 and its pea orthologue exist in an abundant, previously unrecognized soluble form, and partition between cytosol-containing soluble fractions and the chloroplast outer membrane. We show that soluble atToc159 binds directly to the cytosolic domain of atToc33 in a homotypic interaction, contributing to the integration of atToc159 into the chloroplast outer membrane. The data suggest that the function of the Toc complex involves switching of atToc159 between a soluble and an integral membrane form.

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SUMOylation contributes to proteostasis of the chloroplast protein import receptor TOC159 during early development

10.1101/2020.07.12.198945 ◽

2020 ◽

Author(s):

Sonia Accossato ◽

Felix Kessler ◽

Venkatasalam Shanmugabalaji

Keyword(s):

Plant Development ◽

Protein Import ◽

Ubiquitin Proteasome System ◽

Stress Conditions ◽

Chloroplast Biogenesis ◽

Wild Type ◽

Sumoylation Site ◽

Albino Plants ◽

Ubiquitin Proteasome ◽

Import Receptor

AbstractChloroplast biogenesis describes the transition of non-photosynthetic proplastids to photosynthetically active chloroplasts in the cells of germinating seeds. Chloroplast biogenesis requires the import of thousands of nuclear-encoded preproteins and depends on the essential import receptor TOC159, mutation of which results in non-photosynthetic albino plants. We previously showed that ubiquitin-proteasome system (UPS)-dependent regulation of TOC159 levels contributes to the regulation of chloroplast biogenesis during early plant development. Here, we demonstrate that the SUMO (Small Ubiquitin-related Modifier) pathway crosstalks with the ubiquitin-proteasome pathway to affect TOC159 stability during early plant development. We identified a SUMO3-interacting motif (SIM) in the TOC159 GTPase (G-) domain and a SUMO3 covalent SUMOylation site in the membrane (M-) domain. A single K to R substitution (K1370R) in the M-domain disables SUMOylation. Expression of the TOC159K1370R mutant in the toc159 mutant (ppi2) complemented the albino phenotype. Compared to wild type TOC159, TOC159K1370R was destabilized under UPS-inducing stress conditions. However, TOC159K1370R recovered to same protein level as wild type TOC159 in the presence of a proteasome inhibitor. Thus, SUMOylation partially stabilizes TOC159 against UPS-dependent degradation under stress conditions. Our data contribute to the evolving model of tightly controlled proteostasis of the TOC159 import receptor during proplastid to chloroplast transition.

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SUMOylation contributes to proteostasis of the chloroplast protein import receptor TOC159 during early development

eLife ◽

10.7554/elife.60968 ◽

2020 ◽

Vol 9 ◽

Author(s):

Sonia Accossato ◽

Felix Kessler ◽

Venkatasalam Shanmugabalaji

Keyword(s):

Protein Import ◽

Stress Conditions ◽

Chloroplast Biogenesis ◽

Wild Type ◽

Sumoylation Site ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Evolving Model ◽

Germinating Seeds ◽

Import Receptor

Chloroplast biogenesis describes the transition of non-photosynthetic proplastids to photosynthetically active chloroplasts in the cells of germinating seeds. Chloroplast biogenesis requires the import of thousands of nuclear-encoded preproteins by essential import receptor TOC159. We demonstrate that the small ubiquitin-related modifier (SUMO) pathway crosstalks with the ubiquitin–proteasome pathway to affect TOC159 stability during early plant development. We identified a SUMO3-interacting motif (SIM) in the TOC159 GTPase domain and a SUMO3 covalent SUMOylation site in the membrane domain. A single K to R substitution (K1370R) in the M-domain disables SUMOylation. Compared to wild-type TOC159, TOC159K1370R was destabilized under UPS-inducing stress conditions. However, TOC159K1370R recovered to same protein level as wild-type TOC159 in the presence of a proteasome inhibitor. Thus, SUMOylation partially stabilizes TOC159 against UPS-dependent degradation under stress conditions. Our data contribute to the evolving model of tightly controlled proteostasis of the TOC159 import receptor during proplastid to chloroplast transition.

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Faculty Opinions recommendation of Unusual nucleotide-binding properties of the chloroplast protein import receptor, atToc33.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1014228.193161 ◽

2003 ◽

Author(s):

Steven Theg

Keyword(s):

Protein Import ◽

Nucleotide Binding ◽

Binding Properties ◽

Chloroplast Protein Import ◽

Chloroplast Protein ◽

Import Receptor

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Peroxisome Senescence in Human Fibroblasts

Molecular Biology of the Cell ◽

10.1091/mbc.e02-06-0322 ◽

2002 ◽

Vol 13 (12) ◽

pp. 4243-4255 ◽

Cited By ~ 109

Author(s):

Julie E. Legakis ◽

Jay I. Koepke ◽

Chris Jedeszko ◽

Ferdous Barlaskar ◽

Laura J. Terlecky ◽

...

Keyword(s):

Molecular Mechanisms ◽

Protein Import ◽

Human Fibroblasts ◽

Toxic Metabolite ◽

Age Related ◽

Peroxisomal Targeting ◽

Targeting Signal ◽

Effects Of Aging ◽

Antioxidant Enzyme Catalase ◽

Import Receptor

The molecular mechanisms of peroxisome biogenesis have begun to emerge; in contrast, relatively little is known about how the organelle functions as cells age. In this report, we characterize age-related changes in peroxisomes of human cells. We show that aging compromises peroxisomal targeting signal 1 (PTS1) protein import, affecting in particular the critical antioxidant enzyme catalase. The number and appearance of peroxisomes are altered in these cells, and the organelles accumulate the PTS1-import receptor, Pex5p, on their membranes. Concomitantly, cells produce increasing amounts of the toxic metabolite hydrogen peroxide, and we present evidence that this increased load of reactive oxygen species may further reduce peroxisomal protein import and exacerbate the effects of aging.

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Structural Basis of Presequence Recognition by the Mitochondrial Protein Import Receptor Tom20

Cell ◽

10.1016/s0092-8674(00)80691-1 ◽

2000 ◽

Vol 100 (5) ◽

pp. 551-560 ◽

Cited By ~ 352

Author(s):

Yoshito Abe ◽

Toshihiro Shodai ◽

Takanori Muto ◽

Katsuyoshi Mihara ◽

Hisayoshi Torii ◽

...

Keyword(s):

Protein Import ◽

Mitochondrial Protein ◽

Structural Basis ◽

Mitochondrial Protein Import ◽

Import Receptor

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Inactivation of the Neurospora crassa gene encoding the mitochondrial protein import receptor MOM19 by the technique of "sheltered RIP".

Genetics ◽

10.1093/genetics/136.1.107 ◽

1994 ◽

Vol 136 (1) ◽

pp. 107-118 ◽

Cited By ~ 5

Author(s):

T A Harkness ◽

R L Metzenberg ◽

H Schneider ◽

R Lill ◽

W Neupert ◽

...

Keyword(s):

Neurospora Crassa ◽

Target Gene ◽

Protein Import ◽

Selectable Marker ◽

Mitochondrial Protein ◽

Mutant Gene ◽

Mitochondrial Protein Import ◽

Gene Encoding ◽

Precursor Proteins ◽

Import Receptor

Abstract We have used a technique referred to as "sheltered RIP" (repeat induced point mutation) to create mutants of the mom-19 gene of Neurospora crassa, which encodes an import receptor for nuclear encoded mitochondrial precursor proteins. Sheltered RIP permits the isolation of a mutant gene in one nucleus, even if that gene is essential for the survival of the organism, by sheltering the nucleus carrying the mutant gene in a heterokaryon with an unaffected nucleus. Furthermore, the nucleus harboring the RIPed gene contains a selectable marker so that it is possible to shift nuclear ratios in the heterokaryons to a state in which the nucleus containing the RIPed gene predominates in cultures grown under selective conditions. This results in a condition where the target gene product should be present at very suboptimal levels and allows the study of the mutant phenotype. One allele of mom-19 generated by this method contains 44 transitions resulting in 18 amino acid substitutions. When the heterokaryon containing this allele was grown under conditions favoring the RIPed nucleus, no MOM19 protein was detectable in the mitochondria of the strain. Homokaryotic strains containing the RIPed allele exhibit a complex and extremely slow growth phenotype suggesting that the product of the mom-19 gene is important in N. crassa.

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Role of Temperature Stress on Chloroplast Biogenesis and Protein Import in Pea

PLANT PHYSIOLOGY ◽

10.1104/pp.109.137265 ◽

2009 ◽

Vol 150 (2) ◽

pp. 1050-1061 ◽

Cited By ~ 79

Author(s):

Siddhartha Dutta ◽

Sasmita Mohanty ◽

Baishnab C. Tripathy

Keyword(s):

Temperature Stress ◽

Protein Import ◽

Chloroplast Biogenesis

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