More Membranes, more Proteins: Complex Protein Import Mechanisms into Secondary Plastids

Nup153 is a large (153 kD) O-linked glyco-protein which is a component of the basket structure located on the nucleoplasmic face of nuclear pore complexes. This protein exhibits a tripartite structure consisting of a zinc finger domain flanked by large (60-70 kD) NH2- and COOH-terminal domains. When full-length human Nup153 is expressed in BHK cells, it accumulates appropriately at the nucleoplasmic face of the nuclear envelope. Targeting information for Nup153 resides in the NH2-terminal domain since this region of the molecule can direct an ordinarily cytoplasmic protein, pyruvate kinase, to the nuclear face of the nuclear pore complex. Overexpression of Nup153 results in the dramatic accumulation of nuclear poly (A)+ RNA, suggesting an inhibition of RNA export from the nucleus. This is not due to a general decline in nucleocytoplasmic transport or to occlusion or loss of nuclear pore complexes since nuclear protein import is unaffected. While overexpression of certain Nup153 constructs was found to result in the formation of unusual intranuclear membrane arrays, this structural phenotype could not be correlated with the effects on poly (A)+ RNA distribution. The RNA trafficking defect was, however, dependent upon the Nup153 COOH-terminal domain which contains most of the XFXFG repeats. It is proposed that this region of Nup153, lying within the distal ring of the nuclear basket, represents a docking site for mRNA molecules exiting the nucleus.

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GTP hydrolysis by Ran occurs at the nuclear pore complex in an early step of protein import.

The Journal of Cell Biology ◽

10.1083/jcb.131.3.571 ◽

1995 ◽

Vol 131 (3) ◽

pp. 571-581 ◽

Cited By ~ 87

Author(s):

F Melchior ◽

T Guan ◽

N Yokoyama ◽

T Nishimoto ◽

L Gerace

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Import ◽

Protein Import ◽

Small Gtpase ◽

Nuclear Pore ◽

Binding Studies ◽

Early Step ◽

Permeabilized Cells ◽

Complex Protein ◽

Ran Gtp

Mediated import of proteins into the nucleus involves multiple cytosolic factors, including the small GTPase Ran. Whether Ran functions by interacting with other cytosolic proteins or components of the nuclear pore complex has been unclear. Furthermore, the precise transport step where Ran acts has not been determined. To address these questions, we have analyzed the binding interactions of Ran using permeabilized cells and isolated nuclear envelopes. By light and electron microscope immunolocalization, we have found that Ran accumulates specifically at the cytoplasmic surface of the nuclear pore complex when nuclear import in permeabilized cells is inhibited by nonhydrolyzable analogs of GTP. Ran associates with a peripheral pore complex region that is similar to the area where transport ligands accumulate by depletion of ATP, which arrests an early step of transport. Binding studies with isolated nuclear envelopes in the absence of added cytosol indicate that Ran-GTP directly interacts with a pore complex protein. Using blot overlay techniques, we detected a single prominent polypeptide of isolated nuclear envelopes that binds Ran-GTP. This corresponds to the 358-kD protein RanBP2, a Ran binding pore complex protein recently identified by two-hybrid screening. Thus, RanBP2 is likely to constitute the Ran-GTP-binding site detected at the cytoplasmic periphery of the pore complex. These data support a model in which initial ligand binding to the nuclear pore complex occurs at or near RanBP2, and that hydrolysis of GTP by Ran at this site serves to define commitment to the nuclear import pathway.

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Genome-Based Reconstruction of the Protein Import Machinery in the Secondary Plastid of a Chlorarachniophyte Alga

Eukaryotic Cell ◽

10.1128/ec.05264-11 ◽

2012 ◽

Vol 11 (3) ◽

pp. 324-333 ◽

Cited By ~ 30

Author(s):

Yoshihisa Hirakawa ◽

Fabien Burki ◽

Patrick J. Keeling

Keyword(s):

Protein Import ◽

Intermembrane Space ◽

Content Type ◽

Green Algal ◽

Secondary Plastid ◽

Plastid Proteins ◽

Red Algal ◽

Plastid Protein ◽

Secondary Plastids ◽

Envelope Membranes

ABSTRACT Most plastid proteins are encoded by their nuclear genomes and need to be targeted across multiple envelope membranes. In vascular plants, the translocons at the outer and inner envelope membranes of chloroplasts (TOC and TIC, respectively) facilitate transport across the two plastid membranes. In contrast, several algal groups harbor more complex plastids, the so-called secondary plastids, which are surrounded by three or four membranes, but the plastid protein import machinery (in particular, how proteins cross the membrane corresponding to the secondary endosymbiont plasma membrane) remains unexplored in many of these algae. To reconstruct the putative protein import machinery of a secondary plastid, we used the chlorarachniophyte alga Bigelowiella natans , whose plastid is bounded by four membranes and still possesses a relict nucleus of a green algal endosymbiont (the nucleomorph) in the intermembrane space. We identified nine homologs of plant-like TOC/TIC components in the recently sequenced B. natans nuclear genome, adding to the two that remain in the nucleomorph genome ( B. natans TOC75 [BnTOC75] and BnTIC20). All of these proteins were predicted to be localized to the plastid and might function in the inner two membranes. We also show that the homologs of a protein, Der1, that is known to mediate transport across the second membrane in the several lineages with secondary plastids of red algal origin is not associated with plastid protein targeting in B. natans . How plastid proteins cross this membrane remains a mystery, but it is clear that the protein transport machinery of chlorarachniophyte plastids differs from that of red algal secondary plastids.

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Distribution of the SELMA Translocon in Secondary Plastids of Red Algal Origin and Predicted Uncoupling of Ubiquitin-Dependent Translocation from Degradation

Eukaryotic Cell ◽

10.1128/ec.00183-12 ◽

2012 ◽

Vol 11 (12) ◽

pp. 1472-1481 ◽

Cited By ~ 46

Author(s):

Simone Stork ◽

Daniel Moog ◽

Jude M. Przyborski ◽

Ilka Wilhelmi ◽

Stefan Zauner ◽

...

Keyword(s):

Phaeodactylum Tricornutum ◽

Protein Import ◽

Content Type ◽

Evolutionary Origins ◽

Red Algal ◽

Multistep Process ◽

Core Components ◽

Secondary Plastids ◽

Periplastidal Compartment

ABSTRACT Protein import into complex plastids of red algal origin is a multistep process including translocons of different evolutionary origins. The s ymbiont-derived E RAD- l ike ma chinery (SELMA), shown to be of red algal origin, is proposed to be the transport system for preprotein import across the periplastidal membrane of heterokontophytes, haptophytes, cryptophytes, and apicomplexans. In contrast to the canonical endoplasmic reticulum-associated degradation (ERAD) system, SELMA translocation is suggested to be uncoupled from proteasomal degradation. We investigated the distribution of known and newly identified SELMA components in organisms with complex plastids of red algal origin by intensive data mining, thereby defining a set of core components present in all examined organisms. These include putative pore-forming components, a ubiquitylation machinery, as well as a Cdc48 complex. Furthermore, the set of known 20S proteasomal components in the periplastidal compartment (PPC) of diatoms was expanded. These newly identified putative SELMA components, as well as proteasomal subunits, were in vivo localized as PPC proteins in the diatom Phaeodactylum tricornutum . The presented data allow us to speculate about the specific features of SELMA translocation in contrast to the canonical ERAD system, especially the uncoupling of translocation from degradation.

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Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62.

The Journal of Cell Biology ◽

10.1083/jcb.129.4.925 ◽

1995 ◽

Vol 129 (4) ◽

pp. 925-937 ◽

Cited By ~ 257

Author(s):

B M Paschal ◽

L Gerace

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Import ◽

Protein Import ◽

Multicomponent System ◽

Nuclear Pore ◽

Transport Activity ◽

Complex Protein ◽

Multistep Process ◽

Cytosolic Factor ◽

Cytosolic Factors

Protein import into the nucleus is a multistep process that requires the activities of several cytosolic factors. In this study we have purified a cytosolic factor that interacts with the nuclear pore complex glycoprotein p62. Isolation involved biochemical complementation of cytosol depleted of this activity by preadsorption with recombinant p62 and the use of a novel flow cytometry-based assay for quantitation of nuclear import. The purified activity (NTF2) is an apparent dimer of approximately 14-kD subunits and is present at approximately 10(6) copies per cell. We obtained a cDNA encoding NTF2 and showed that the recombinant protein restores transport activity to p62-pretreated cytosol. Our data suggest that NTF2 acts at a relatively late stage of nuclear protein import, subsequent to the initial docking of nuclear import ligand at the nuclear envelope. NTF2 interacts with at least one additional cytosolic transport activity, indicating that it could be part of a multicomponent system of cytosolic factors that assemble at the pore complex during nuclear import.

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In Silico Tools for the Prediction of Protein Import into Secondary Plastids

Methods in Molecular Biology - Plastids ◽

10.1007/978-1-4939-8654-5_25 ◽

2018 ◽

pp. 381-394 ◽

Cited By ~ 1

Author(s):

Daniel Moog

Keyword(s):

In Silico ◽

Protein Import ◽

Secondary Plastids ◽

In Silico Tools

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Genetic and physical interactions between Srp1p and nuclear pore complex proteins Nup1p and Nup2p.

The Journal of Cell Biology ◽

10.1083/jcb.126.3.619 ◽

1994 ◽

Vol 126 (3) ◽

pp. 619-630 ◽

Cited By ~ 70

Author(s):

K D Belanger ◽

M A Kenna ◽

S Wei ◽

L I Davis

Keyword(s):

Nuclear Envelope ◽

Nuclear Pore Complex ◽

Protein Import ◽

Sequence Similarity ◽

Nuclear Architecture ◽

Nuclear Pore ◽

Beta Catenin ◽

Complex Protein ◽

Oligomeric Complex ◽

Physical Interactions

Nup1p is a yeast nuclear pore complex protein (nucleoporin) required for nuclear protein import, mRNA export and maintenance of normal nuclear architecture. We have used a genetic approach to identify other proteins that interact functionally with Nup1p. Here we describe the isolation of seventeen mutants that confer a requirement for Nup1p in a background in which this protein is normally not essential. Some of the mutants require wild-type Nup1p, while others are viable in combination with specific nup1 alleles. Several of the mutants show nonallelic noncomplementation, suggesting that the products may be part of a hetero-oligomeric complex. One is allelic to srp1 which, although it was identified in an unrelated screen, was shown to encode a protein that is localized to the nuclear envelope (Yano, R., M. Oakes, M. Yamaghishi, J. A. Dodd, and M. Nomura. 1992. Mol. Cell. Biol. 12:5640-5651). We have used immunoprecipitation and fusion protein precipitation to show that Srp1p forms distinct complexes with both Nup1p and the related nucleoporin Nup2p, indicating that Srp1p is a component of the nuclear pore complex. The distant sequence similarity between Srp1p and the beta-catenin/desmoplakin family, coupled with the altered structure of the nuclear envelope in nup1 mutants, suggests that Srp1p may function in attachment of the nuclear pore complex to an underlying nuclear skeleton.

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