scholarly journals Tail-anchored PEX26 targets peroxisomes via a PEX19-dependent and TRC40-independent class I pathway

2013 ◽  
Vol 200 (5) ◽  
pp. 651-666 ◽  
Author(s):  
Yuichi Yagita ◽  
Takahide Hiromasa ◽  
Yukio Fujiki
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Adenosine Triphosphatase ◽  
Transmembrane Domain ◽  
Peroxisomal Membrane ◽  
Basic Amino Acids ◽  
C Terminus ◽  
Peroxisomal Targeting ◽  
Ta Proteins

Tail-anchored (TA) proteins are anchored into cellular membranes by a single transmembrane domain (TMD) close to the C terminus. Although the targeting of TA proteins to peroxisomes is dependent on PEX19, the mechanistic details of PEX19-dependent targeting and the signal that directs TA proteins to peroxisomes have remained elusive, particularly in mammals. The present study shows that PEX19 formed a complex with the peroxisomal TA protein PEX26 in the cytosol and translocated it directly to peroxisomes by interacting with the peroxisomal membrane protein PEX3. Unlike in yeast, the adenosine triphosphatase TRC40, which delivers TA proteins to the endoplasmic reticulum, was dispensable for the peroxisomal targeting of PEX26. Moreover, the basic amino acids within the luminal domain of PEX26 were essential for binding to PEX19 and thereby for peroxisomal targeting. Finally, our results suggest that a TMD that escapes capture by TRC40 and is followed by a highly basic luminal domain directs TA proteins to peroxisomes via the PEX19-dependent route.

scholarly journals Biosynthesis of the dystonia-associated AAA+ ATPase torsinA at the endoplasmic reticulum

2006 ◽  
Vol 401 (2) ◽  
pp. 607-612 ◽  
Author(s):  
Anna C. Callan ◽  
Sandra Bunning ◽  
Owen T. Jones ◽  
Stephen High ◽  
Eileithyia Swanton
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Integral Membrane Protein ◽  
Signal Peptidase ◽  
Aaa Atpase ◽  
C Terminus ◽  
Membrane Spanning ◽  
Aaa Atpases

TorsinA is a widely expressed AAA+ (ATPases associated with various cellular activities) ATPase of unknown function. Previous studies have described torsinA as a type II protein with a cleavable signal sequence, a single membrane spanning domain, and its C-terminus located in the ER (endoplasmic reticulum) lumen. However, in the present study we show that torsinA is not in fact an integral membrane protein. Instead we find that the mature protein associates peripherally with the ER membrane, most likely through an interaction with an integral membrane protein. Consistent with this model, we provide evidence that the signal peptidase complex cleaves the signal sequence of torsinA, and we show that the region previously suggested to form a transmembrane domain is translocated into the lumen of the ER. The finding that torsinA is a peripheral, and not an integral membrane protein as previously thought, has important implications for understanding the function of this novel ATPase.

scholarly journals Targeting of a Tail-anchored Protein to Endoplasmic Reticulum and Mitochondrial Outer Membrane by Independent but Competing Pathways

2001 ◽  
Vol 12 (8) ◽  
pp. 2482-2496 ◽  
Author(s):  
Nica Borgese ◽  
Ilaria Gazzoni ◽  
Massimo Barberi ◽  
Sara Colombo ◽  
Emanuela Pedrazzini
Keyword(s):  
Endoplasmic Reticulum ◽  
Outer Membrane ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Mitochondrial Outer Membrane ◽  
C Terminus ◽  
Green Fluorescent ◽  
Ta Proteins

Many mitochondrial outer membrane (MOM) proteins have a transmembrane domain near the C terminus and an N-terminal cytosolic moiety. It is not clear how these tail-anchored (TA) proteins posttranslationally select their target, but C-terminal charged residues play an important role. To investigate how discrimination between MOM and endoplasmic reticulum (ER) occurs, we used mammalian cytochrome b 5, a TA protein existing in two, MOM or ER localized, versions. Substitution of the seven C-terminal residues of the ER isoform or of green fluorescent protein reporter constructs with one or two arginines resulted in MOM-targeted proteins, whereas a single C-terminal threonine caused promiscuous localization. To investigate whether targeting to MOM occurs from the cytosol or after transit through the ER, we tagged a MOM-directed construct with a C-terminal N-glycosylation sequence. Although in vitro this construct was efficiently glycosylated by microsomes, the protein expressed in vivo localized almost exclusively to MOM, and was nearly completely unglycosylated. The small fraction of glycosylated protein was in the ER and was not a precursor to the unglycosylated form. Thus, targeting occurs directly from the cytosol. Moreover, ER and MOM compete for the same polypeptide, explaining the dual localization of some TA proteins.

scholarly journals Pex24 and Pex32 are required to tether peroxisomes to the ER for organelle biogenesis, positioning and segregation in yeast

2020 ◽  
Vol 133 (16) ◽  
pp. jcs246983 ◽  
Author(s):  
Fei Wu ◽  
Rinse de Boer ◽  
Arjen M. Krikken ◽  
Arman Akşit ◽  
Nicola Bordin ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Matrix Protein ◽  
Protein Import ◽  
Hansenula Polymorpha ◽  
Major Effect ◽  
Organelle Biogenesis ◽  
Peroxisomal Membrane ◽  
Contact Sites ◽  
Membrane Growth

ABSTRACTThe yeast Hansenula polymorpha contains four members of the Pex23 family of peroxins, which characteristically contain a DysF domain. Here we show that all four H. polymorpha Pex23 family proteins localize to the endoplasmic reticulum (ER). Pex24 and Pex32, but not Pex23 and Pex29, predominantly accumulate at peroxisome–ER contacts. Upon deletion of PEX24 or PEX32 – and to a much lesser extent, of PEX23 or PEX29 – peroxisome–ER contacts are lost, concomitant with defects in peroxisomal matrix protein import, membrane growth, and organelle proliferation, positioning and segregation. These defects are suppressed by the introduction of an artificial peroxisome–ER tether, indicating that Pex24 and Pex32 contribute to tethering of peroxisomes to the ER. Accumulation of Pex32 at these contact sites is lost in cells lacking the peroxisomal membrane protein Pex11, in conjunction with disruption of the contacts. This indicates that Pex11 contributes to Pex32-dependent peroxisome–ER contact formation. The absence of Pex32 has no major effect on pre-peroxisomal vesicles that occur in pex3 atg1 deletion cells.

scholarly journals Peroxisome assembly factor 1: nonsense mutation in a peroxisome-deficient Chinese hamster ovary cell mutant and deletion analysis.

1994 ◽  
Vol 14 (8) ◽  
pp. 5458-5465 ◽  
Author(s):  
T Tsukamoto ◽  
N Shimozawa ◽  
Y Fujiki
Keyword(s):  
Amino Acids ◽  
Chinese Hamster Ovary ◽  
Nonsense Mutation ◽  
Chinese Hamster ◽  
General Rule ◽  
Ovary Cell ◽  
Cell Mutant ◽  
Peroxisomal Membrane ◽  
C Terminus ◽  
Assembly Factor

A cDNA encoding 35-kDa peroxisome assembly factor 1 (PAF-1), a peroxisomal integral membrane protein, was cloned from Chinese hamster ovary (CHO) cells and sequenced. The CHO PAF-1 comprised 304 amino acids, one residue shorter than rat or human PAF-1, and showed high homology to rat and human PAF-1: 90 and 86% at the nucleotide sequence level and 92 and 90% in amino acid sequence, respectively. PAF-1 from these three species contains a conserved cysteine-rich sequence at the C-terminal region which is exactly the same as that of a novel cysteine-rich RING finger motif family. PAF-1 cDNA from a peroxisome-deficient CHO cell mutant, Z65 (T. Tsukamoto, S. Yokota, and Y. Fujiki, J. Cell Biol. 110:651-660, 1990), contained a nonsense mutation at the codon for Trp-114, resulting in premature termination. Truncation in PAF-1 of either 19 amino acids from the N terminus or 92 residues from the C terminus maintained the peroxisome assembly-restoring activity when tested in both the Z65 mutant and the fibroblasts from a Zellweger patient. In contrast, deletion of 27 or 102 residues from the N or C terminus eliminated the activity. PAF-1 is encoded by free polysomal RNA, consistent with a general rule for biogenesis of peroxisomal proteins, including membrane polypeptides, implying the posttranslational transport and integration of PAF-1 into peroxisomal membrane.

scholarly journals Regulation of Bestrophin Cl Channels by Calcium: Role of the C Terminus

2008 ◽  
Vol 132 (6) ◽  
pp. 681-692 ◽  
Author(s):  
Qinghuan Xiao ◽  
Andrew Prussia ◽  
Kuai Yu ◽  
Yuan-yuan Cui ◽  
H. Criss Hartzell
Keyword(s):  
Amino Acids ◽  
Density Functional ◽  
Transmembrane Domain ◽  
Channel Gating ◽  
Regulatory Domain ◽  
Acidic Amino Acids ◽  
C Terminus ◽  
Ef Hand ◽  
Cl Channels

Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of Cl− ion channels that are activated by intracellular Ca2+. At present, the structures and mechanisms responsible for Ca2+ sensing remain unknown. Here, we have used a combination of molecular modeling, density functional–binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in Ca2+ sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential for Ca2+ sensing by hBest1. The cluster of five amino acids (293–308) is crucial for normal channel gating by Ca2+ because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by Ca2+. Using homology models built on the crystal structure of calmodulin (CaM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind Ca2+ with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative Ca2+-binding loop (312–323) reduced the apparent Ca2+ affinity by 20-fold. In addition, the D312G and D323N mutations abolished Ca2+-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350–390) that is required for Ca2+ activation and plays a role in current rundown. These experiments identify a region of hBest1 (312–323) that is involved in the gating of hBest1 by Ca2+ and suggest a model in which Ca2+ binding to EF1 activates the channel in a process that requires the acidic domain (293–308) and another regulatory domain (350–390). Many of the ∼100 disease-causing mutations in hBest1 are located in this region that we have implicated in Ca2+ sensing, suggesting that these mutations disrupt hBest1 channel gating by Ca2+.

scholarly journals Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida

2001 ◽  
Vol 69 (12) ◽  
pp. 7839-7850 ◽  
Author(s):  
Gillian D. Pullinger ◽  
R. Sowdhamini ◽  
Alistair J. Lax
Keyword(s):  
Amino Acids ◽  
Fusion Proteins ◽  
Pasteurella Multocida ◽  
Transmembrane Domain ◽  
Polyclonal Antibodies ◽  
Full Length ◽  
Cell Binding ◽  
Terminal Fragment ◽  
C Terminus ◽  
N Terminus

ABSTRACT The locations of the catalytic and receptor-binding domains of thePasteurella multocida toxin (PMT) were investigated. N- and C-terminal fragments of PMT were cloned and expressed as fusion proteins with affinity tags. Purified fusion proteins were assessed in suitable assays for catalytic activity and cell-binding ability. A C-terminal fragment (amino acids 681 to 1285) was catalytically active. When microinjected into quiescent Swiss 3T3 cells, it induced changes in cell morphology typical of toxin-treated cells and stimulated DNA synthesis. An N-terminal fragment with a His tag at the C terminus (amino acids 1 to 506) competed with full-length toxin for binding to surface receptors and therefore contains the cell-binding domain. The inactive mutant containing a mutation near the C terminus (C1165S) also bound to cells in this assay. Polyclonal antibodies raised to the N-terminal PMT region bound efficiently to full-length native toxin, suggesting that the N terminus is surface located. Antibodies to the C terminus of PMT were microinjected into cells and inhibited the activity of toxin added subsequently to the medium, confirming that the C terminus contains the active site. Analysis of the PMT sequence predicted a putative transmembrane domain with predicted hydrophobic and amphipathic helices near the N terminus over the region of homology to the cytotoxic necrotizing factors. The C-terminal end of PMT was predicted to be a mixed α/β domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.

scholarly journals The SH3 domain of the Saccharomyces cerevisiae peroxisomal membrane protein Pex13p functions as a docking site for Pex5p, a mobile receptor for the import PTS1-containing proteins.

1996 ◽  
Vol 135 (1) ◽  
pp. 97-109 ◽  
Author(s):  
Y Elgersma ◽  
L Kwast ◽  
A Klein ◽  
T Voorn-Brouwer ◽  
M van den Berg ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Protein ◽  
Protein Import ◽  
Sh3 Domain ◽  
Type I ◽  
Peroxisomal Membrane ◽  
Peroxisomal Membrane Protein ◽  
Peroxisomal Targeting ◽  
Src Homology

We identified a Saccharomyces cerevisiae peroxisomal membrane protein, Pex13p, that is essential for protein import. A point mutation in the COOH-terminal Src homology 3 (SH3) domain of Pex13p inactivated the protein but did not affect its membrane targeting. A two-hybrid screen with the SH3 domain of Pex13p identified Pex5p, a receptor for proteins with a type I peroxisomal targeting signal (PTS1), as its ligand. Pex13p SH3 interacted specifically with Pex5p in vitro. We determined, furthermore, that Pex5p was mainly present in the cytosol and only a small fraction was associated with peroxisomes. We therefore propose that Pex13p is a component of the peroxisomal protein import machinery onto which the mobile Pex5p receptor docks for the delivery of the selected PTS1 protein.

scholarly journals The peroxin Pex17p of the yeast Yarrowia lipolytica is associated peripherally with the peroxisomal membrane and is required for the import of a subset of matrix proteins.

1997 ◽  
Vol 17 (5) ◽  
pp. 2511-2520 ◽  
Author(s):  
J J Smith ◽  
R K Szilard ◽  
M Marelli ◽  
R A Rachubinski
Keyword(s):  
Amino Acids ◽  
Oleic Acid ◽  
Yarrowia Lipolytica ◽  
Matrix Proteins ◽  
Peroxisomal Membrane ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Carboxyl Terminal ◽  
Peroxisomal Targeting Signal ◽  
Yeast Yarrowia Lipolytica

PEX genes encode peroxins, which are required for the biogenesis of peroxisomes. The Yarrowia lipolytica PEX17 gene encodes the peroxin Pex17p, which is 671 amino acids in length and has a predicted molecular mass of 75,588 Da. Pex17p is peripherally associated with the peroxisomal membrane. The carboxyl-terminal tripeptide, Gly-Thr-Leu, of Pex17p is not necessary for its targeting to peroxisomes. Synthesis of Pex17p is low in cells grown in glucose-containing medium and increases after the cells are shifted to oleic acid-containing medium. Cells of the pex17-1 mutant, the original mutant strain, and the pex17-KA mutant, a strain in which most of the PEX17 gene is deleted, fail to form normal peroxisomes but instead contain numerous large, multimembraned structures. The import of peroxisomal matrix proteins in these mutants is selectively impaired. This selective import is not a function of the nature of the peroxisomal targeting signal. We suggest a regulatory role for Pex17p in the import of a subset of matrix proteins into peroxisomes.

scholarly journals A Cold-Sensitive Listeria monocytogenes Mutant Has a Transposon Insertion in a Gene Encoding a Putative Membrane Protein and Shows Altered (p)ppGpp Levels

2006 ◽  
Vol 72 (6) ◽  
pp. 3955-3959 ◽  
Author(s):  
Siqing Liu ◽  
Darrell O. Bayles ◽  
Tricia M. Mason ◽  
Brian J. Wilkinson
Keyword(s):  
Listeria Monocytogenes ◽  
Low Temperature ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Critical Role ◽  
Peptide Sequence ◽  
Temperature Adaptation ◽  
Signal Peptide Sequence ◽  
C Terminus ◽  
Cold Sensitive

ABSTRACT A cold-sensitive Listeria monocytogenes mutant designated cld-14 was obtained by transposon Tn917 mutagenesis. The gene interrupted by Tn917 in cld-14 was the L. monocytogenes LMOf2365_1485 homolog, which exhibits 45.7% homology to the Bacillus subtilis yqfF locus. LMOf2365_1485, here designated pgpH, encodes a putative integral membrane protein with a predicted molecular mass of 81 kDa. PgpH is predicted to contain a conserved N-terminal signal peptide sequence, seven transmembrane helices, and a hydrophilic C terminus, which likely extends into the cytosol. The Tn917 insertion in pgpH is predicted to result in production of a premature polypeptide truncated at the fifth transmembrane domain. The C terminus of PgpH, which is probably absent in cld-14, contains a highly conserved HD domain that belongs to a metal-dependent phosphohydrolase family. Strain cld-14 accumulated higher levels of (p)ppGpp than the wild type accumulated, indicating that the function of PgpH may be to adjust cellular (p)ppGpp levels during low-temperature growth. The cld-14pgpH + complemented strain was able to grow at a low temperature, like the parent strain, providing direct evidence that the activity of PgpH is important in low-temperature adaptation. Because of its predicted membrane location, PgpH may play a critical role in sensing the environmental temperature and altering cellular (p)ppGpp levels to allow the organism to adapt to low temperatures.

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