Abstract
Alix/AIP1 is a proline-rich protein that has been implicated in apoptosis, endocytic membrane trafficking and viral budding. To further elucidate the functions of Alix, we used RNA interference to specifically suppress its expression. Depletion of Alix caused a striking redistribution of early endosomes from a peripheral to a perinuclear location. The redistribution of endosomes did not affect transferrin recycling or degradation of endocytosed epidermal growth factor receptors, although the uptake of transferrin was mildly reduced when Alix was downregulated. Quantitative immunoelectron microscopy showed that multivesicular endosomes of Alix-depleted cells contained normal amounts of CD63, whereas their levels of lysobisphosphatidic acid were reduced. Alix depletion also caused an accumulation of unusual actin structures that contained clathrin and cortactin, a protein that couples membrane dynamics to the cortical actin cytoskeleton. Our results suggest that Alix functions in the actin-dependent intracellular positioning of endosomes, but that it is not essential for endocytic recycling or for trafficking of membrane proteins between early and late endosomes in non-polarised cells.
Highlights
Endocytosis is essential for constitutive uptake of proteinbound nutrients and for ligand-induced receptor downregulation
Internalised molecules are delivered to early endosomes, where activated growth factor receptors are sorted into late endosomes and lysosomes for degradation while nutrient receptors are recycled to the plasma membrane (Gruenberg, 2001)
Immunoblot analysis showed that small interfering RNA (siRNA) oligos against Alix caused the strongest depletion of the protein levels after 144 hours (72 hours in the presence of siRNA followed by 72 hours in the absence of RNA, Fig. 1A)
Summary
Endocytosis is essential for constitutive uptake of proteinbound nutrients and for ligand-induced receptor downregulation. Internalised molecules are delivered to early endosomes, where activated growth factor receptors are sorted into late endosomes and lysosomes for degradation while nutrient receptors are recycled to the plasma membrane (Gruenberg, 2001). The molecular machineries that mediate endocytosis and endosomal sorting are currently being characterized; here we have studied the functions of a protein that has been implicated in the regulation of protein trafficking through endosomes. MVBs mediate the transport between early endosomes and late endosomes/lysosomes. This pathway is required for a number of biological processes, such as growth factor receptor downregulation, antigen presentation and retroviral budding. It is thought to interact with ubiquitinated membrane proteins and to deliver these further to the so-called endosomal sorting complexes required for transport (ESCRTs)
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