scholarly journals Essential role of the G-domain in targeting of the protein import receptor atToc159 to the chloroplast outer membrane

2002 ◽  
Vol 159 (5) ◽  
pp. 845-854 ◽  
Author(s):  
Jörg Bauer ◽  
Andreas Hiltbrunner ◽  
Petra Weibel ◽  
Pierre-Alexandre Vidi ◽  
Mayte Alvarez-Huerta ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Essential Role ◽  
Protein Import ◽  
Chloroplast Envelope ◽  
Chloroplast Biogenesis ◽  
Gtp Binding ◽  
Precursor Proteins ◽  
Albino Mutant ◽  
Import Receptor

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.

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

2001 ◽  
Vol 154 (2) ◽  
pp. 309-316 ◽  
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.

scholarly journals Dual role of the receptor Tom20 in specificity and efficiency of protein import into mitochondria

2010 ◽  
Vol 108 (1) ◽  
pp. 91-96 ◽  
Author(s):  
Hayashi Yamamoto ◽  
Nobuka Itoh ◽  
Shin Kawano ◽  
Yoh-ichi Yatsukawa ◽  
Takaki Momose ◽  
...  
Keyword(s):  
Essential Role ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Dual Role ◽  
Mitochondrial Protein Import ◽  
Targeting Signal ◽  
Terminal Element ◽  
Import Receptor

Mitochondria import most of their resident proteins from the cytosol, and the import receptor Tom20 of the outer-membrane translocator TOM40 complex plays an essential role in specificity of mitochondrial protein import. Here we analyzed the effects of Tom20 binding on NMR spectra of a long mitochondrial presequence and found that it contains two distinct Tom20-binding elements. In vitro import and cross-linking experiments revealed that, although the N-terminal Tom20-binding element is essential for targeting to mitochondria, the C-terminal element increases efficiency of protein import in the step prior to translocation across the inner membrane. Therefore Tom20 has a dual role in protein import into mitochondria: recognition of the targeting signal in the presequence and tethering the presequence to the TOM40 complex to increase import efficiency.

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

Genetics ◽  
1994 ◽  
Vol 136 (1) ◽  
pp. 107-118 ◽  
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.

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

2009 ◽  
Vol 150 (2) ◽  
pp. 1050-1061 ◽  
Author(s):  
Siddhartha Dutta ◽  
Sasmita Mohanty ◽  
Baishnab C. Tripathy
Keyword(s):  
Temperature Stress ◽  
Protein Import ◽  
Chloroplast Biogenesis

scholarly journals Functional Relevance of the Transmembrane Domain and Cytoplasmic Tail of the Pseudorabies Virus Glycoprotein H for Membrane Fusion

2018 ◽  
Vol 92 (12) ◽  
Author(s):  
Melina Vallbracht ◽  
Walter Fuchs ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter
Keyword(s):  
Membrane Fusion ◽  
Fusion Proteins ◽  
Essential Role ◽  
Pseudorabies Virus ◽  
Transmembrane Domain ◽  
Cytoplasmic Tail ◽  
Class Iii ◽  
Complex Needs ◽  
Hsv 1

ABSTRACTHerpesvirus membrane fusion depends on the core fusion machinery, comprised of glycoproteins B (gB) and gH/gL. Although gB structurally resembles autonomous class III fusion proteins, it strictly depends on gH/gL to drive membrane fusion. Whether the gH/gL complex needs to be membrane anchored to fulfill its function and which role the gH cytoplasmic (CD) and transmembrane domains (TMD) play in fusion is unclear. While the gH CD and TMD play an important role during infection, soluble gH/gL of herpes simplex virus 1 (HSV-1) seems to be sufficient to mediate cell-cell fusion in transient assays, arguing against an essential contribution of the CD and TMD. To shed more light on this apparent discrepancy, we investigated the role of the CD and TMD of the related alphaherpesvirus pseudorabies virus (PrV) gH. For this purpose, we expressed C-terminally truncated and soluble gH and replaced the TMD with a glycosylphosphatidylinositol (gpi) anchor. We also generated chimeras containing the TMD and/or CD of PrV gD or HSV-1 gH. Proteins were characterized in cell-based fusion assays and during virus infection. Although truncation of the CD resulted in decreased membrane fusion activity, the mutant proteins still supported replication of gH-negative PrV, indicating that the PrV gH CD is dispensable for viral replication. In contrast, PrV gH lacking the TMD, membrane-anchored via a lipid linker, or comprising the PrV gD TMD were nonfunctional, highlighting the essential role of the gH TMD for function. Interestingly, despite low sequence identity, the HSV-1 gH TMD could substitute for the PrV gH TMD, pointing to functional conservation.IMPORTANCEEnveloped viruses depend on membrane fusion for virus entry. While this process can be mediated by only one or two proteins, herpesviruses depend on the concerted action of at least three different glycoproteins. Although gB has features of bona fide fusion proteins, it depends on gH and its complex partner, gL, for fusion. Whether gH/gL prevents premature fusion or actively triggers gB-mediated fusion is unclear, and there are contradictory results on whether gH/gL function requires stable membrane anchorage or whether the ectodomains alone are sufficient. Our results show that in pseudorabies virus gH, the transmembrane anchor plays an essential role for gB-mediated fusion while the cytoplasmic tail is not strictly required.

scholarly journals Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope.

1996 ◽  
Vol 134 (2) ◽  
pp. 315-327 ◽  
Author(s):  
Y Ma ◽  
A Kouranov ◽  
S E LaSala ◽  
D J Schnell
Keyword(s):  
Outer Membrane ◽  
Protein Import ◽  
Small Subunit ◽  
Chloroplast Envelope ◽  
Stable Complex ◽  
Nucleoside Triphosphate ◽  
Cross Linking ◽  
Outer Envelope ◽  
Bisphosphate Carboxylase ◽  
Precursor Proteins

The interactions of precursor proteins with components of the chloroplast envelope were investigated during the early stages of protein import using a chemical cross-linking strategy. In the absence of energy, two components of the outer envelope import machinery, IAP86 and IAP75, cross-linked to the transit sequence of the precursor to the small subunit of ribulose-1, 5-bisphosphate carboxylase (pS) in a precursor binding assay. In the presence of concentrations of ATP or GTP that support maximal precursor binding to the envelope, cross-linking to the transit sequence occurred predominantly with IAP75 and a previously unidentified 21-kD polypeptide of the inner membrane, indicating that the transit sequence had inserted across the outer membrane. Cross-linking of envelope components to sequences in the mature portion of a second precursor, preferredoxin, was detected in the presence of ATP or GTP, suggesting that sequences distant from the transit sequence were brought into the vicinity of the outer membrane under these conditions. IAP75 and a third import component, IAP34, were coimmunoprecipitated with IAP86 antibodies from solubilized envelope membranes, indicating that these three proteins form a stable complex in the outer membrane. On the basis of these observations, we propose that IAP86 and IAP75 act as components of a multisubunit complex to mediate energy-independent recognition of the transit sequence and subsequent nucleoside triphosphate-induced insertion of the transit sequence across the outer membrane.

scholarly journals The major chloroplast envelope polypeptide is the phosphate translocator and not the protein import receptor

Nature ◽  
1991 ◽  
Vol 353 (6342) ◽  
pp. 364-367 ◽  
Author(s):  
Ulf-Ingo Flügge ◽  
Andreas Weber ◽  
Karsten Fischer ◽  
Fritz Lottspeich ◽  
Christoph Eckerskorn ◽  
...  
Keyword(s):  
Protein Import ◽  
Chloroplast Envelope ◽  
Phosphate Translocator ◽  
Import Receptor

scholarly journals Increased levels of the mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol

2021 ◽  
Author(s):  
Anna M. Schlagowski ◽  
Katharina Knöringer ◽  
Sandrine Morlot ◽  
Ana Sáchez Vicente ◽  
Felix Boos ◽  
...  
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Model System ◽  
Model Systems ◽  
Aggregate Formation ◽  
Mitochondrial Protein Import ◽  
Precursor Proteins ◽  
Polyq Protein ◽  
Rate Limiting

AbstractThe formation of protein aggregates is a hallmark of neurodegenerative diseases. Observations on patient material and model systems demonstrated links between aggregate formation and declining mitochondrial functionality, but the causalities remained unclear. We used yeast as model system to analyze the relevance of mitochondrial processes for the behavior of an aggregation-prone polyQ protein derived from human huntingtin. Induction of Q97-GFP rapidly leads to insoluble cytosolic aggregates and cell death. Although this aggregation impairs mitochondrial respiration only slightly, it interferes with efficient import of mitochondrial precursor proteins. Mutants in the import component Mia40 are hypersensitive to Q97-GFP. Even more surprisingly, Mia40 overexpression strongly suppresses the formation of toxic Q97-GFP aggregates both in yeast and in human cells. Based on these observations, we propose that the posttranslational import into mitochondria competes with aggregation-prone cytosolic proteins for chaperones and proteasome capacity. Owing to its rate-limiting role for mitochondrial protein import, Mia40 acts as a regulatory component in this competition. This role of Mia40 as dynamic regulator in mitochondrial biogenesis can apparently be exploited to stabilize cytosolic proteostasis. (174/175 words)

scholarly journals A crucial role of the mitochondrial protein import receptor MOM19 for the biogenesis of mitochondria

1994 ◽  
Vol 124 (5) ◽  
pp. 637-648 ◽  
Author(s):  
TA Harkness ◽  
FE Nargang ◽  
I van der Klei ◽  
W Neupert ◽  
R Lill
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Mitochondrial Outer Membrane ◽  
Growth Defect ◽  
The Novel ◽  
Genetic Method ◽  
Severe Growth Defect ◽  
Biogenesis Of Mitochondria ◽  
Import Receptor

The novel genetic method of "sheltered RIP" (repeat induced point mutation) was used to generate a Neurospora crassa mutant in which MOM19, a component of the protein import machinery of the mitochondrial outer membrane, can be depleted. Deficiency in MOM19 resulted in a severe growth defect, but the cells remained viable. The number of mitochondrial profiles was not grossly changed, but mutant mitochondria were highly deficient in cristae membranes, cytochromes, and protein synthesis activity. Protein import into isolated mutant mitochondria was decreased by factors of 6 to 30 for most proteins from all suborganellar compartments. Proteins like the ADP/ATP carrier, MOM19, and cytochrome c, whose import into wild-type mitochondria occurs independently of MOM19 became imported normally showing that the reduced import activities are solely caused by a lack of MOM19. Depletion of MOM19 reveals a close functional relationship between MOM19 and MOM22, since loss of MOM19 led to decreased levels of MOM22 and reduced protein import through MOM22. Furthermore, MOM72 does not function as a general backup receptor for MOM19 suggesting that these two proteins have distinct precursor specificities. These findings demonstrate that the import receptor MOM19 fulfills an important role in the biogenesis of mitochondria and that it is essential for the formation of mitochondria competent in respiration and phosphorylation.

scholarly journals Essential Role of VIPP1 in Chloroplast Envelope Maintenance in Arabidopsis

2012 ◽  
Vol 24 (9) ◽  
pp. 3695-3707 ◽  
Author(s):  
Lingang Zhang ◽  
Yusuke Kato ◽  
Stephanie Otters ◽  
Ute C. Vothknecht ◽  
Wataru Sakamoto
Keyword(s):  
Essential Role ◽  
Chloroplast Envelope
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