scholarly journals In vitro insertion of the 22-kD peroxisomal membrane protein into isolated rat liver peroxisomes.

1993 ◽  
Vol 123 (6) ◽  
pp. 1717-1725 ◽  
Author(s):  
P Diestelkötter ◽  
W W Just
Keyword(s):  
Membrane Protein ◽  
Rat Liver ◽  
Atp Hydrolysis ◽  
Firefly Luciferase ◽  
In Vitro Translation ◽  
Membrane Insertion ◽  
Peroxisomal Membrane ◽  
Permeabilized Cells ◽  
Peroxisomal Membrane Protein

The membrane insertion of the 22-kD integral peroxisomal membrane protein (PMP 22) was studied in a system in which peroxisomes isolated from rat liver were incubated with the [35S]methionine-labeled in vitro translation product of PMP 22 mRNA. Membrane insertion of PMP 22 was demonstrated by protease treatment of peroxisomes in the absence and presence of detergent. Approximately 35% of total in vitro translated PMP 22 became protease resistant after a 1-h incubation at 26 degrees C. Import was dependent on time and temperature, did not require ATP or GTP and was not inhibited by N-ethylmaleimide treatment of neither the soluble components of the translation mixture nor of the isolated peroxisomes. In contrast to these results it was recently shown that the import of the peroxisomal marker, firefly luciferase, into peroxisomes of permeabilized cells was dependent on ATP hydrolysis and was blocked by N-ethylmaleimide pretreatment of the cytosol-depleted cells (Rapp et al., 1993; Wendland and Subramani, 1993). Therefore, the present data suggest that insertion of PMP 22 into the peroxisomal membrane and translocation of firefly luciferase into peroxisomes follow distinct mechanisms. At low temperature binding of PMP 22 to the peroxisomal membrane was not influenced whereas insertion was strongly inhibited. Pretreatment of peroxisomes with subtilisin reduced binding to a low level and completely abolished insertion. Therefore it is suggested that binding is prerequisite to insertion and that insertion may be mediated by a proteinaceous receptor.

Sorting of the 70-kDa Peroxisomal Membrane Protein into Rat Liver Peroxisomes in Vitro

1996 ◽  
Vol 804 (1 Peroxisomes) ◽  
pp. 663-665 ◽  
Author(s):  
TSUNEO IMANAKA ◽  
TATSUYA TAKANO ◽  
TAKASHI OSUMI ◽  
TAKASHI HASHIMOTO
Keyword(s):  
Membrane Protein ◽  
Rat Liver ◽  
Peroxisomal Membrane ◽  
Peroxisomal Membrane Protein ◽  
Liver Peroxisomes

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 membrane remodeling protein Pex11p activates the GTPase Dnm1p during peroxisomal fission

2015 ◽  
Vol 112 (20) ◽  
pp. 6377-6382 ◽  
Author(s):  
Chris Williams ◽  
Lukasz Opalinski ◽  
Christiane Landgraf ◽  
Joseph Costello ◽  
Michael Schrader ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Crucial Role ◽  
Initial Phase ◽  
Membrane Remodeling ◽  
Gtpase Activating Protein ◽  
Peroxisomal Membrane ◽  
Additional Function ◽  
Peroxisomal Membrane Protein

The initial phase of peroxisomal fission requires the peroxisomal membrane protein Peroxin 11 (Pex11p), which remodels the membrane, resulting in organelle elongation. Here, we identify an additional function for Pex11p, demonstrating that Pex11p also plays a crucial role in the final step of peroxisomal fission: dynamin-like protein (DLP)-mediated membrane scission. First, we demonstrate that yeast Pex11p is necessary for the function of the GTPase Dynamin-related 1 (Dnm1p) in vivo. In addition, our data indicate that Pex11p physically interacts with Dnm1p and that inhibiting this interaction compromises peroxisomal fission. Finally, we demonstrate that Pex11p functions as a GTPase activating protein (GAP) for Dnm1p in vitro. Similar observations were made for mammalian Pex11β and the corresponding DLP Drp1, indicating that DLP activation by Pex11p is conserved. Our work identifies a previously unknown requirement for a GAP in DLP function.

scholarly journals Hydrophobic Regions Adjacent to Transmembrane Domains 1 and 5 Are Important for the Targeting of the 70-kDa Peroxisomal Membrane Protein

2007 ◽  
Vol 282 (46) ◽  
pp. 33831-33844 ◽  
Author(s):  
Yoshinori Kashiwayama ◽  
Kota Asahina ◽  
Masashi Morita ◽  
Tsuneo Imanaka
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Intracellular Localization ◽  
Chinese Hamster ◽  
Transmembrane Domains ◽  
Peroxisomal Membrane ◽  
Peroxisomal Membrane Protein

The 70-kDa peroxisomal membrane protein (PMP70) is a major component of peroxisomal membranes. Human PMP70 consists of 659 amino acid residues and has six putative transmembrane domains (TMDs). PMP70 is synthesized on cytoplasmic ribosomes and targeted posttranslationally to peroxisomes by an unidentified peroxisomal membrane protein targeting signal (mPTS). In this study, to examine the mPTS within PMP70 precisely, we expressed various COOH-terminally or NH2-terminally deleted constructs of PMP70 fused with green fluorescent protein (GFP) in Chinese hamster ovary cells and determined their intracellular localization by immunofluorescence. In the COOH-terminally truncated PMP70, PMP70(AA.1-144)-GFP, including TMD1 and TMD2 of PMP70, was still localized to peroxisomes. However, by further removal of TMD2, PMP70(AA.1-124)-GFP lost the targeting ability, and PMP70(TMD2)-GFP did not target to peroxisomes by itself. The substitution of TMD2 in PMP70(AA.1-144)-GFP for TMD4 or TMD6 did not affect the peroxisomal localization, suggesting that PMP70(AA.1-124) contains the mPTS and an additional TMD is required for the insertion into the peroxisomal membrane. In the NH2-terminal 124-amino acid region, PMP70 possesses hydrophobic segments in the region adjacent to TMD1. By the disruption of these hydrophobic motifs by the mutation of L21Q/L22Q/L23Q or I70N/L71Q, PMP70(AA.1-144)-GFP lost targeting efficiency. The NH2-terminally truncated PMP70, GFP-PMP70(AA.263-375), including TMD5 and TMD6, exhibited the peroxisomal localization. PMP70(AA.263-375) also possesses hydrophobic residues (Ile307/Leu308) in the region adjacent to TMD5, which were important for targeting. These results suggest that PMP70 possesses two distinct targeting signals, and hydrophobic regions adjacent to the first TMD of each region are important for targeting.

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