Journal: The FEBS journal

Article Title: Membrane topologies of PEX13 and PEX14 provide new insights on the mechanism of protein import into peroxisomes.

doi: 10.1111/febs.14697

Figure Lengend Snippet: Figure 1 – Schematic representation of PEX13 and PEX14 and their interactions with each other and with PEX5. The amino acid positions of the globular domains of Rattus Norvegicus PEX13 and PEX14 proteins are indicated (CC, Coiled coil; NTD, N-terminal domain of PEX14; SH3, Src Homology 3 domain). Putative transmembrane domains (TMDs; light blue cylinders) were predicted with PRALINE™ [83] and PHOBIUS [84] using an alignment of twenty protein sequences representative of eukaryotic evolution. The pentapeptide motifs of PEX5 [43] are represented in orange. Black lines indicate the epitopes recognized by some of the antibodies used in this work. The interactions between PEX13, PEX14 and PEX5 are indicated as red (yeast) or blue (mammals) arrows. The SH3 domain of yeast PEX13 interacts with a PXXP motif (residues 87-90) located between the NTD and TMD of PEX14 [61,85]. In mammals, the SH3 domain of PEX13 interacts with the NTD of PEX14 [41,63]. The region comprising amino acid residues 236-246 of yeast PEX13, which corresponds to a region in the mammalian peroxin located between TMD2 and TMD3, interacts with an undefined region (marked with “?”) of PEX14 [47]. The SH3 of PEX13 also interacts with the N-terminal half of PEX5 in yeast [36,37,85]. A similar interaction involving residues 219-403 of human PEX13 (which includes the SH3 domain) and the N-terminal half of PEX5 has been described [38]. Residues 1-135 of mammalian PEX13 were also reported to interact with pentapeptides motifs 2-4 of PEX5 [45]. The NTD of PEX14 interacts with pentapeptide motifs present in the N-terminal half of PEX5 both in mammals and yeast/fungi [43,45,49,71,86]. A C- terminal domain of yeast PEX14 (residues 235-341) has been shown to interact with PEX5 [49,71], an interaction involving the second pentapeptide motif of PEX5 [49] (Reviewed in [51,57,70]).

Article Snippet: The following antibodies were used: rabbit antibody against full-length human PEX14 protein [14], rabbit antibody against amino acid residues 327-377 of human PEX14 protein (Bethyl laboratories.

Techniques:

Journal: The FEBS journal

Article Title: Membrane topologies of PEX13 and PEX14 provide new insights on the mechanism of protein import into peroxisomes.

doi: 10.1111/febs.14697

Figure Lengend Snippet: Figure 2 – PEX14 is a transmembrane protein with a Nin-Cout topology. A- Expression and purification of H6TEVPEX14 using IMAC. Culture aliquots collected before (lane NI) and after (lane I) protein induction, lysed cell extract (lane T) and the corresponding soluble (lane S) and insoluble protein fractions (lane P), the non-bound protein fraction from the Nickel-NTA beads (NB) and the purified protein () – H6TEVPEX14 (lane P14) were analyzed by SDS-PAGE, blotted onto a nitrocellulose membrane, and stained with Ponceau S. B- Proteoliposomes (Lipos) containing either PEX14 (left panel) or H6TEVPEX14 (H6PEX14; right panel) were treated or not with PK (400 µg/mL) in the presence or absence of TX-100, and analyzed by SDS-PAGE/Western blot. PEX14 was detected with antibodies against the full-length (FL) protein. Equivalent amounts of recombinant proteins (Lanes I; 130 ng) and the corresponding reconstituted proteins were loaded onto the gels. C- As in B, but using PEX14 that was subjected to the reconstitution protocol in the absence of lipids. D- As in B, but using antibodies directed to the full-length protein, the C-terminal residues 327-377 of human PEX14, and the N-terminal histidine-tag. E- PEX14 proteoliposomes were treated with PK (400 µg/mL) or trypsin (Try, 400 µg/mL) and analyzed by SDS-PAGE/Western blot using an antibody against the full-length protein. The ~46-kDa PEX14 fragment obtained after trypsin digestion is recognized by the anti-PEX14(327-377) antibody (data not shown). Identical results were obtained with H6TEVPEX14; in this case, the two fragments with ~20 and ~30 kDa are recognized by the anti-His antibody (data not shown), suggesting that the 30-kDa fragment is a partial proteolysis product. F- Proteoliposomes, PK-treated proteoliposomes, and PK alone were subjected to SDS- PAGE, blotted onto a Sequi-blot PVDF membrane and stained with Coomassie Brilliant Blue. PK- resistant fragments of PEX14 (, 40 kDa and ~20 kDa) and protein bands derived from PK itself (*)

Article Snippet: The following antibodies were used: rabbit antibody against full-length human PEX14 protein [14], rabbit antibody against amino acid residues 327-377 of human PEX14 protein (Bethyl laboratories.

Techniques: Expressing, Purification, SDS Page, Membrane, Staining, Western Blot, Recombinant, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Homologue NME3 of DYNAMO1 Regulates Peroxisome Division

doi: 10.3390/ijms21218040

Figure Lengend Snippet: Aberrant morphology of peroxisomes in NME3-deficient fibroblasts. ( A – C ) peroxisomes in the fibroblasts from a healthy control (upper panels) and a patient F741 (lower panels) carrying a homozygous mutation in the initiation codon of NME3 were visualized by indirect immunofluorescent staining with antibodies to catalase ( A ), PTS1 ( B ), ADAPS ( C ) and Pex14 ( A – C ). Insets show the images of the boxed areas. Bars, 20 µm and 2 µm (insets). Elongated peroxisomes are frequently observed in the patient-derived fibroblasts. ( D ) proteolytic processing of AOx, thiolase, and ADAPS was accessed by immunoblotting of cell-lysates of fibroblasts from a healthy control and the patient with antibodies to AOx, ADAPS, thiolase, Pex3, and LDHA, respectively. LDHA was used as a loading control. ( E ) histogram of peroxisome length measured in three each fibroblasts from a healthy control (1400 peroxisomes in three cells) and a patient F741 (1038 peroxisomes in three cells).

Article Snippet: Rabbit antibodies to acyl-CoA oxidase (AOx) [ ], 3-ketoacyl-CoA thiolase [ ], ADAPS [ ], PTS1 peptide [ ], Pex3 [ ], Pex14 [ ], Mff [ ], DYNAMO1 [ ], 70-kDa peroxisomal membrane protein (PMP70) [ ], and guinea pig anti-Pex14 antibody [ ] were as described.

Techniques: Control, Mutagenesis, Staining, Derivative Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Homologue NME3 of DYNAMO1 Regulates Peroxisome Division

doi: 10.3390/ijms21218040

Figure Lengend Snippet: Intracellular localization of NME3-HA 2 in HeLa cells. ( A ) NME3-HA 2 was expressed in HeLa cells. NME3-HA 2 was stained with rabbit anti-HA antibody (green). Peroxisomes and mitochondria were visualized with guinea pig anti-Pex14 (upper panels) and mouse anti-Tom20 (lower panels) antibodies, respectively. Peroxisomes are shown by a pseudo-color image. Scale bar, 10 µm. Higher magnification images of the boxed regions were shown (Inset). Scale bar, 5 µm. Arrowheads indicate peroxisomal, not mitochondrial, localization of NME3-HA 2 . ( B ) immunoblotting of mock- (-) and NME3-HA 2 - (+) transfected HeLa cells. Approximately ten times more of total proteins were loaded in lanes 1 and 4 than those in lanes 2 and 3. NME3-HA 2 was detected with antibodies to HA (upper panel, lanes 1 and 2) and NME3 (ABclonal) (upper panel, lanes 3 and 4). Two bands (solid and open arrowheads) were detected and termed NME3-1-HA 2 and NME3-2-HA 2 , respectively, by the expression of NME3-HA 2 . Dots indicate non-specific bands. β-actin, a loading control.

Article Snippet: Rabbit antibodies to acyl-CoA oxidase (AOx) [ ], 3-ketoacyl-CoA thiolase [ ], ADAPS [ ], PTS1 peptide [ ], Pex3 [ ], Pex14 [ ], Mff [ ], DYNAMO1 [ ], 70-kDa peroxisomal membrane protein (PMP70) [ ], and guinea pig anti-Pex14 antibody [ ] were as described.

Techniques: Staining, Western Blot, Transfection, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Homologue NME3 of DYNAMO1 Regulates Peroxisome Division

doi: 10.3390/ijms21218040

Figure Lengend Snippet: Intracellular localization of non-tagged NME3 in HeLa cells. Non-tagged NME3 was expressed in HeLa cells and stained with the antibody raised to DYNAMO1 (green). Peroxisomes and mitochondria were visualized with guinea pig anti-Pex14 ( upper panels ) and mouse anti-Tom20 ( lower panels ) antibodies, respectively. Peroxisomes are shown by a pseudo-color image. Scale bar, 10 µm. Higher magnification images of the boxed regions were shown (Inset). Scale bar, 5 µm. Arrowheads and arrows indicate peroxisomal and mitochondrial localization of NME3, respectively.

Article Snippet: Rabbit antibodies to acyl-CoA oxidase (AOx) [ ], 3-ketoacyl-CoA thiolase [ ], ADAPS [ ], PTS1 peptide [ ], Pex3 [ ], Pex14 [ ], Mff [ ], DYNAMO1 [ ], 70-kDa peroxisomal membrane protein (PMP70) [ ], and guinea pig anti-Pex14 antibody [ ] were as described.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Homologue NME3 of DYNAMO1 Regulates Peroxisome Division

doi: 10.3390/ijms21218040

Figure Lengend Snippet: NME3 is localized to peroxisomes and involved in the fission of peroxisomes. ( A ) intracellular localization of endogenous NME3 in HeLa cells was assessed by indirect immunofluorescent cell staining with antibodies to DYNAMO1 (green), Pex14, and Tom20 as in . Scale bar, 10 µm. Higher magnification images of the boxed regions are shown (Inset). Scale bar, 5 µm. Note that mitochondrial localization of NME3 is distinct (lower panels). ( B ) HeLa cells transfected with dsRNA against NME3 (#28, see ) were verified by indirect immunofluorescent cell staining with anti-bodies to DYNAMO1 and Tom20. Scale bar, 10 µm. ( C ) HeLa cells transfected with dsRNA against ATAD1 were verified by indirect immunofluorescent cell staining as in ( A ). Scale bar, 10 µm. Higher magnification images of the boxed regions are shown (Inset). Scale bar, 5 µm. Note that localization of NME3 in peroxisomes (arrowheads), but not mitochondria, is more readily discernible. ( D ) HeLa cells co-transfected with a set of two dsRNAs against ATAD1 and NME3 were verified by indirect immuno- fluorescent cell staining with antibodies to DYNAMO1 and Pex14. Higher magnification images of the boxed regions are shown (Inset). Note that peroxisomes are frequently elongated. Scale bar, 10 µm. Higher magnification images of the boxed regions were shown (Inset). Scale bar, 5 µm. ( E ) relative fluorescent intensity of NME3 in HeLa cells transfected with mock (-, n = 38) or dsRNA against ATAD1 (+, n = 25) was quantified. * p < 0.05. ( F , G ) transcription level of ATAD1 ( F ) and NME3 ( G ) in HeLa cells treated as in ( C ) was quantified by quantitative real-time PCR (n = 3). ( H ) protein level of Pex14 was verified by immunoblotting (left). β-actin, a loading control. Protein level of Pex14 was represented as values relative to that in mock-treated HeLa cells (right, n = 3). ( I ) the number of peroxisomes in HeLa cells untreated (n = 37), transfected with dsRNA against ATAD1 alone (n = 37), or a set of ATAD1 and NME3 (n = 33) was represented. *** p < 0.001, by Tukey-Kramer test. n.s., not significant. ( J ) NME3 level is elevated by ATAD1 knockdown. siControl and siATAD1 were separately transfected twice with a 24 h interval to HeLa cells that had been transfected for 6 h with a plasmid encoding NME3. After 24-h cell culture, transcription level of ATAD1 (left) was quantified by quantitative real-time PCR (n = 2). Center, NME3 expression levels were assessed by western blotting of respective cell lysates with anti-NME3C antibody. Two bands, NME3-1 and NME3-2, marked by solid and open arrowheads were detected. β-actin, a loading control. Dots indicate non-specific bands; right, NME3-1 and NME3-2 bands were quantified and represented by taking as 1 NME3-1 in siControl -transfected cells in lane 1. ( K ) upper row, NME3 was expressed in PEX11β −/− mouse embryonic fibroblasts (MEF) and its intracellular localization was verified by staining with antibodies to DYNAMO1 ( a ), DLP1 ( b ), and Pex14 ( c ). Scale bar, 10 µm. Higher magnification images of the boxed regions were shown (Inset). Scale bar, 5 µm. Note that NME3 was detected in the limited area of an elongated peroxisome (arrowhead). Panel ( e ), signal intensity of NME3, DLP1, and Pex14 in the elongated peroxisome indicated with its length in the merged view was analyzed by line scanning and represented. Note that NME3 was localized at the DLP1-accumulated potential constriction site (arrowhead in ( a – d )) where signal of Pex14 is weak and the both sides adjacent to this region the constriction site.

Article Snippet: Rabbit antibodies to acyl-CoA oxidase (AOx) [ ], 3-ketoacyl-CoA thiolase [ ], ADAPS [ ], PTS1 peptide [ ], Pex3 [ ], Pex14 [ ], Mff [ ], DYNAMO1 [ ], 70-kDa peroxisomal membrane protein (PMP70) [ ], and guinea pig anti-Pex14 antibody [ ] were as described.

Techniques: Staining, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Control, Knockdown, Plasmid Preparation, Cell Culture, Expressing