CONICET | Buscador de Institutos y Recursos Humanos

The Ca2+ pumps that deplete the cytosol of Ca2+ ions are crucial to Ca2+ homeostasis. The Ca2+ pump from the plasma membrane (PMCA) is an essential component of animal cells. We have recently shown that functional PMCA (human isoform 4xb) can be constitutively produced in the yeast S. cerevisiae (Cura et. al., 2008). Here we used fluorescence confocal microscopy to investigate the localization of a PMCA-GFP fusion in the yeast cell. Most of the expressed protein was observed associated with perinuclear endoplasmic reticulum membranes and at the cell periphery in the plasma membrane or just underneath it. The distribution of fluorescence intensity at the cell cortex exhibited a marked patchy pattern indicating that the PMCA-GFP was concentrated in discrete clusters while excluded from other membrane domains. After photobleaching of a limited region of the cell cortex, the initial fluorescence recovered rapidly (<5 min) regenerating the PMCA clusters at exactly the same location. To study the dynamics of cortical PMCA-GFP, we used fluorescence correlation spectroscopy (FCS). These experiments showed that the protein follows anomalous diffusion in this membrane probably due to interactions with other components of the membrane. The fully active PMCAct120-GFP mutant ct120 lacking the C-terminal 120 residues showed a distribution pattern similar to the PMCA-GFP. Thus, it seems that the PMCA clustering is neither mediated by interactions via the C-terminal PDZ binding domain nor by self-association through the C-terminal regulatory region.