Spatial dynamics of receptor-mediated endocytic trafficking in budding yeast revealed by using fluorescent α-factor derivatives

Abstract

Much progress defining the order and timing of endocytic internalization events has come as a result of real-time, live-cell fluorescence microscopy. Although the availability of numerous endocytic mutants makes yeast an especially valuable organism for functional analysis of endocytic dynamics, a serious limitation has been the lack of a fluorescent cargo for receptor-mediated endocytosis. We have now synthesized biologically active fluorescent mating-pheromone derivatives and demonstrated that receptormediated endocytosis in budding yeast occurs via the clathrin-and actin-mediated endocytosis pathway. We found that endocytic proteins first assemble into patches on the plasma membrane, and then ␣-factor associates with the patches. Internalization occurs next, concomitant with actin assembly at patches. Additionally, endocytic vesicles move toward early endosomes on actin cables. Early endosomes also associate with actin cables, and they actively move toward endocytic sites to capture vesicles being released from the plasma membrane. Thus, endocytic vesicle formation and capture of the newly released vesicles by early endosomes occur in a highly concerted manner, mediated by the actin cytoskeleton.actin ͉ cytoskeleton ͉ endocytosis ͉ endosome

Endocytic Accessory Factors and Regulation of Clathrin-Mediated Endocytosis

“…However, an elegant experiment by Schmid and coworkers showed that local clustering of biotinylated TfR into tetramers using fluorescent streptavidin did promote CCP nucleation, suggesting that localized concentration of signaling motifs promoted CCP nucleation by recruiting adaptor proteins (Liu et al 2010). The picture in yeast cells is less clearly defined and, unlike mammalian cells, the arrival of the receptor cargo, afactor apparently occurred after the initiation of bud nucleation (Toshima et al 2006).…”

Cell-cycle regulation of formin-mediated actin cable assembly

“…Yeast strains used in this study are listed in Table S1. C-terminal GFP and RFP tags were integrated by homologous recombination, as described previously (77,104). All strains were grown at 30°C in standard rich medium (Yeast Extract Peptone Dextrose, YPD) or synthetic medium supplemented with appropriate amino acids, unless otherwise noted.…”

Bifurcation of the endocytic pathway into Rab5-dependent and -independent transport to the vacuole

“…The unexpected connection between endocytic trafficking and the AP-3 pathway motivated us to further examine the dynamics of endocytic vesicles and Apl5p-residing endosomes in vps21D ypt52D cells. Abp1p is a marker used for visualizing the formation and internalization of endocytic vesicles 37 , and we previously showed that after binding to a-factor, the Ste2p receptor is recruited to and endocytosed via Abp1p-labelled clathrin-coated vesicles 23,28 . Accordingly, we considered Abp1-mCherry patches to be endocytic vesicles in which a-factor and its receptor would be incorporated, and analysed their dynamics in vps21D ypt52D cells.…”

“…1c,d). We next utilized Alexa Fluor 594-a-factor (A594-a-factor), to label the receptor-mediated endocytic pathway 23,28 and examine the localization of Vps21p in endocytic compartments. GFP-Vps21p was highly colocalized with A594-a-factor-labelled endosomes at 5-10 min after a-factor internalization (Fig.…”

“…We have previously developed fluorescently labelled derivatives of a-factor as novel endocytic markers that bind to the Ste2 receptor, and then become internalized by receptor-mediated endocytosis, finally undergoing trafficking to the vacuole 23 . These fluorescent molecules label the endocytic pathway specifically and time dependently, and do not label other pathways such as the recycling pathway, thereby allowing visualization of the whole route taken by endocytosed cargo before delivery to the vacuole.…”