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Structural/functional molecular organization of peroxisomal proteins
Author(s) -
Makkar Randhir Singh,
Contreras Miguel A,
Smith Brian,
Haq Ehtishamul,
Paintlia Ajaib Singh,
Singh Inderjit
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a96-c
Subject(s) - peroxisome , biochemistry , membrane protein , catalase , transmembrane domain , chemistry , atp binding cassette transporter , cytosol , transmembrane protein , peroxisomal targeting signal , biology , enzyme , membrane , receptor , transporter , gene
The objective of this work was to investigate the structural/functional organization of membrane (adrenoleukodystrophy, ALDP, and very long chain acyl‐CoA synthetase, VLCS) and matrix (catalase) proteins in peroxisomes because loss of their functions results in X‐ALD and Zellweger like‐syndrome, respectively. We studied ALDP‐VLCS interaction using yeast two‐hybrid system (YTHS) and surface plasmon resonance (SPR). YTHS studies demonstrated that the transmembrane domain of ALDP might physically be in contact with VLCS in peroxisomes as compared to N’ or C’‐terminal regions of ALDP. SPR studies document that purified VLCS interact with a domain of internal loop of ALDP on the luminal side of peroxisomal membrane with higher affinity than the same domain carrying a X‐ALDP mutation. Furthermore, catalase, a tetramer that assembles in the cytosol was used to investigate for interactions in the matrix of peroxisomes, using a catalase affinity resin and purified catalase transferred to nitrocellulose membrane after SDS‐PAGE. These approaches identified a catalase interacting protein of 74 kDa from rat liver purified peroxisomes as well as from cytosol. This protein was identified as L‐bifunctional enzyme (L‐BFE) by proteomic analysis. Immunoprecipitation of catalase co‐precipitated L‐BFE, further supporting this result. Interference of L‐BFE expression (siRNA) down regulated the proteins levels of catalase but not its cellular distribution. Taken together, these interactions studies document the structural organization of these proteins in peroxisomes. Supported by grants C06 RR018823, RR015455 and NS22576 from NIH.