Role of Pex19p in the targeting of PMP70 to peroxisome.
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.
Kashiwayama
Yoshinori
Y
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Japan.
Asahina
Kota
K
Shibata
Hiroyuki
H
Morita
Masashi
M
Muntau
Ania C
AC
Roscher
Adelbert A
AA
Wanders
Ronald J A
RJ
Shimozawa
Nobuyuki
N
Sakaguchi
Masao
M
Kato
Hiroaki
H
Imanaka
Tsuneo
T
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001693:Biological Transport, Active; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020675:Peroxisomes / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D012995:Solubility
C063305:ABCD3 protein, human; D018528:ATP-Binding Cassette Transporters; D018076:DNA, Complementary; D008565:Membrane Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C087343:PEX19 protein, human