Live Cell FRET Microscopy

Abstract

The adrenoleukodystrophy protein (ALDP) and the 70-kDa peroxisomal membrane protein (PMP70) are half-ATP-binding cassette (ABC) transporters in the mammalian peroxisome membrane. Mutations in the gene encoding ALDP result in a devastating neurodegenerative disorder, X-linked adrenoleukodystrophy (X-ALD) that is associated with elevated levels of very long chain fatty acids because of impaired peroxisomal ␤-oxidation. The interactions of peroxisomal ABC transporters, their role in the peroxisomal membrane, and their functions in disease pathogenesis are poorly understood. Studies on ABC transporters revealed that half-transporters have to dimerize to gain functionality. So far, conflicting observations are described for ALDP. By the use of in vitro methods (yeast two-hybrid and immunoprecipitation assays) on the one hand, it was shown that ALDP can form homodimers as well as heterodimers with PMP70 and ALDR, while on the other hand, it was demonstrated that ALDP and PMP70 exclusively homodimerize. To circumvent the problems of artificial interactions due to biochemical sample preparation in vitro, we investigated proteinprotein interaction of ALDP in its physiological environment by FRET microscopy in intact living cells. The statistical relevance of FRET data was determined in two different ways using probability distribution shift analysis and Kolmogorov-Smirnov statistics. We demonstrate in vivo that ALDP and PMP70 form homodimers as well as ALDP/PMP70 heterodimers where ALDP homodimers predominate. Using C-terminal deletion constructs of ALDP, we demonstrate that the last 87 C-terminal amino acids harbor the most important protein domain mediating these interactions, and that the N-terminal transmembrane region of ALDP has an additional stabilization effect on ALDP homodimers. Loss of ALDP homo-or heterodimerization is highly relevant for understanding the disease mechanisms of X-ALD.The adrenoleukodystrophy protein (ALDP, 2 ABCD1) is a member of the ATP-binding cassette (ABC) transporter protein family, and one of four ABC transporters in the mammalian peroxisomal membrane. The other members are the adrenoleukodystrophy-related protein (ALDR, ABCD2), the 70-kDa peroxisomal membrane protein (PMP70, ABCD3), and the PMP70-related protein (P70R/PMP69, ABCD4) (1-6).Mutations in the gene encoding ALDP cause X-linked adrenoleukodystrophy (X-ALD), the most common inherited peroxisomal disorder that is characterized by the abnormal accumulation of saturated very long chain fatty acids (VLCFAs) predominantly in brain white matter, adrenal cortex, and testis (7-10). The clinical phenotype is highly variable ranging from the devastating childhood cerebral form with progressive demyelination to the existence of asymptomatic individuals. The pathogenesis of X-ALD and especially the exact function of peroxisomal ABC transporters in the disease process are still completely unknown. ALDP and the other peroxisomal ABC transporters may be involved in the import of VLCFAs or activated fatty acids across the peroxisomal membrane...

Identification of a New Fatty Acid Synthesis-Transport Machinery at the Peroxisomal Membrane

“…The ABC transporter within the complex is composed mainly of ALDP or PMP70 homo-oligomers, which is consistent with reports from earlier studies (16). However, we again confirmed that ALDP also has the capacity to bind PMP70 in vivo.…”

“…2A) showed that ALDP had a significant higher affinity (p Ͻ 0.01) to form homo-oligomers (BRET 50 , 0.85) than heterooligomers with PMP70 (BRET 50 , 2.05). These findings are in line with those obtained by FRET (16). In addition, the data may suggest that PMP70 has a comparable affinity to homo-oligomerize (BRET 50 , 1.22) and is more likely to exist as a homooligomeric than a hetero-oligomeric transporter (this difference was not statistically significant).…”

“…ALDP, PMP70, and ALDR belong to the group of half-transporters, which implies that their functionality depends on dimerization. It was demonstrated previ-ously that ALDP and PMP70 can form homodimers as well as heterodimers with other peroxisomal ABC half-transporters (13)(14)(15)(16). ALDP as a homodimer is able to transport VLCFAs, most probably VLCFA-CoAs, from the cytosol into the peroxisomal lumen for ␤-oxidation, as demonstrated in yeast (17).…”

“…Saturation Experiments-In a previous study, we demonstrated, by means of live cell FRET microscopy in vivo, that ALDP and PMP70 form homodimers as well as ALDP-PMP70 heterodimers, where ALDP homodimers displayed highest values followed by PMP70-PMP70 and ALDP-PMP70, respectively (16). In this study, the interactions of both half-transporters (ALDP and PMP70) with FASN and ACLY were established.…”

Substrate Specificity Overlap and Interaction between Adrenoleukodystrophy Protein (ALDP/ABCD1) and Adrenoleukodystrophy-related Protein (ALDRP/ABCD2)

“…Although a mirror expression pattern is often observed between ALDP and ALDRP when specific cell types are analyzed (9), peroxisomal ABC transporters have overlapping expression patterns rendering possible such interactions (5, 10). Coimmunoprecipitation experiments or FRET analysis have demonstrated heterodimerization in cells overexpressing the peroxisomal ABC transporters (11,12). Although the peroxisomal localization of ALDP, ALDRP, and PMP70 is clearly demonstrated, PMP69 has recently been described to be localized in the endoplasmic reticulum and was found to be absent in the peroxisome.…”