Role of Pex19p in the targeting of PMP70 to peroxisome
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Role of Pex19p in the targeting of PMP70 to peroxisome. / Kashiwayama, Yoshinori; Asahina, Kota; Shibata, Hiroyuki; Morita, Masashi; Muntau, Ania C; Roscher, Adelbert A; Wanders, Ronald J A; Shimozawa, Nobuyuki; Sakaguchi, Masao; Kato, Hiroaki; Imanaka, Tsuneo.
in: Biochim Biophys Acta, Jahrgang 1746, Nr. 2, 15.12.2005, S. 116-28.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Role of Pex19p in the targeting of PMP70 to peroxisome
AU - Kashiwayama, Yoshinori
AU - Asahina, Kota
AU - Shibata, Hiroyuki
AU - Morita, Masashi
AU - Muntau, Ania C
AU - Roscher, Adelbert A
AU - Wanders, Ronald J A
AU - Shimozawa, Nobuyuki
AU - Sakaguchi, Masao
AU - Kato, Hiroaki
AU - Imanaka, Tsuneo
PY - 2005/12/15
Y1 - 2005/12/15
N2 - Pex19p is a protein required for the peroxisomal membrane synthesis. The 70-kDa peroxisomal membrane protein (PMP70) is synthesized on free cytosolic ribosomes and then inserted posttranslationally into peroxisomal membranes. Pex19p has been shown to play an important role in this process. Using an in vitro translation system, we investigated the role of Pex19p as a chaperone and identified the regions of PMP70 required for the interaction with Pex19p. When PMP70 was translated in the presence of purified Pex19p, a large part of PMP70 existed as soluble form and was co-immunoprecipitated with Pex19p. However, in the absence of Pex19p, PMP70 formed aggregates during translation. To identify the regions that interact with Pex19p, various truncated PMP70 were translated in the presence of Pex19p and subjected to co-immunoprecipitation. The interaction was markedly reduced by the deletion of the NH(2)-terminal 61 amino acids or the region around TMD6. Further, we expressed these deletion constructs of PMP70 in fusion with the green fluorescent protein in CHO cells. Fusion proteins lacking these Pex19p binding sites did not display any peroxisomal localization. These results suggest that Pex19p binds to PMP70 co-translationally and keeps PMP70 as a proper conformation for the localization to peroxisome.
AB - Pex19p is a protein required for the peroxisomal membrane synthesis. The 70-kDa peroxisomal membrane protein (PMP70) is synthesized on free cytosolic ribosomes and then inserted posttranslationally into peroxisomal membranes. Pex19p has been shown to play an important role in this process. Using an in vitro translation system, we investigated the role of Pex19p as a chaperone and identified the regions of PMP70 required for the interaction with Pex19p. When PMP70 was translated in the presence of purified Pex19p, a large part of PMP70 existed as soluble form and was co-immunoprecipitated with Pex19p. However, in the absence of Pex19p, PMP70 formed aggregates during translation. To identify the regions that interact with Pex19p, various truncated PMP70 were translated in the presence of Pex19p and subjected to co-immunoprecipitation. The interaction was markedly reduced by the deletion of the NH(2)-terminal 61 amino acids or the region around TMD6. Further, we expressed these deletion constructs of PMP70 in fusion with the green fluorescent protein in CHO cells. Fusion proteins lacking these Pex19p binding sites did not display any peroxisomal localization. These results suggest that Pex19p binds to PMP70 co-translationally and keeps PMP70 as a proper conformation for the localization to peroxisome.
KW - ATP-Binding Cassette Transporters/chemistry
KW - Animals
KW - Base Sequence
KW - Binding Sites
KW - Biological Transport, Active
KW - CHO Cells
KW - Cricetinae
KW - DNA, Complementary/genetics
KW - Green Fluorescent Proteins/genetics
KW - Humans
KW - In Vitro Techniques
KW - Membrane Proteins/chemistry
KW - Mice
KW - Peptide Fragments/chemistry
KW - Peroxisomes/metabolism
KW - Protein Binding
KW - Protein Biosynthesis
KW - RNA, Messenger/genetics
KW - Recombinant Fusion Proteins/chemistry
KW - Sequence Deletion
KW - Solubility
U2 - 10.1016/j.bbamcr.2005.10.006
DO - 10.1016/j.bbamcr.2005.10.006
M3 - SCORING: Journal article
C2 - 16344115
VL - 1746
SP - 116
EP - 128
JO - Biochim Biophys Acta
JF - Biochim Biophys Acta
SN - 0006-3002
IS - 2
ER -