Cell biology and biochemistry of protein transport towards lysosome-related organelles
Zusammenfassung der Projektergebnisse
We purified the recombinant full octameric BLOC-1 complex and elucidated its overall structure to be highly asymmetric, rod-shaped employing the EM negative staining technique. A detailed analysis of this rod revealed eight more globular domains aligned like beads on a string. The overall length was 270 +/- 5 Å and the diameter was 30 Å for the globular domains. Using analytical ultracentrifugation the molecular weight was calculated to be 160 kDa, the sedimentation coefficient to be 4.75 +/- 0.04 S and the frictional ratio f/fo 1.93 +/- 0.03. These data are consistent with the results of the EM analysis and previously published data on the endogenous complex and imply that every subunit is present in the complex only once. We plan to refine our analyses. Moreover, we addressed interactions with the AP-3 complex and found that these are direct, i.e. not involving other factors, and were furthermore able to demonstrate that the membrane-proximal AP-3 core, but not the membrane-distal appendage regions, mediates these interactions. Additionally, we found direct interactions with the melanosomal cargo proteins Tyrosinase, Trp1 and Pmel17 and the SNARE proteins Syntaxin13 and SNAP-25, some of which had already been reported to be mislocalized in BLOC-1 depleted cells. Since these proteins are all integral membrane proteins and have relatively short cytoplasmic domains, these data indicate that the BLOC-1 complex operates in close proximity to the membrane, further highlighted by the additionally identified lipid-binding properties of the recombinant complex. Because AP-3 is involved in sorting of melanosomal cargo proteins and SNARE proteins, we suggest that BLOC-1 might be involved in this sorting as potential regulator. We plan to identify the binding sites of these interactors and submit these data along with the EM structural analysis.
Projektbezogene Publikationen (Auswahl)
- Ang2/fat-free is a conserved subunit of the Golgi-associated retrograde protein complex. Mol. Biol. Cell, 2010 Oct;21(19):3386-95
Pérez-Victoria FJ, Schindler C, Magadán JG, Mardones GA, Delevoye C, Romao M, Raposo G, Bonifacino JS
