Spatial dynamics of receptor-mediated endocytic trafficking in budding yeast revealed by using fluorescent ␣-factor derivatives Junko Y. Toshima*†, Jiro Toshima*†, Marko Kaksonen*, Adam C. Martin*, David S. King‡, and David G. Drubin*§ *Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202; and ‡Howard Hughes Medical Institute, University of California, Berkeley, CA 94720-3202 Communicated by Randy Schekman, University of California, Berkeley, CA, February 7, 2006 (received for review December 24, 2005) Much progress defining the order and timing of endocytic inter- expressed in cells at steady state (9). However, because Ste2-GFP nalization events has come as a result of real-time, live-cell fluo- is not introduced externally to cells and then tracked through the rescence microscopy. Although the availability of numerous endo- endocytic pathway over time, it is not possible to know the identity cytic mutants makes yeast an especially valuable organism for of compartments labeled by Ste2-GFP. This molecule is expected functional analysis of endocytic dynamics, a serious limitation has to label both endocytic and biosynthetic compartments, and po- been the lack of a fluorescent cargo for receptor-mediated endo- tentially nonphysiological compartments if the GFP tag causes cytosis. We have now synthesized biologically active fluorescent missorting of this integral membrane protein. mating-pheromone derivatives and demonstrated that receptor- mediated endocytosis in budding yeast occurs via the clathrin- and Results and Discussion actin-mediated endocytosis pathway. We found that endocytic For our synthesis of fluorescent ␣-factor derivatives, Alexa Fluor- proteins first assemble into patches on the plasma membrane, and 488 C5 or -594 C5 maleimide was conjugated to the ␧-amine of lysine ␣ then -factor associates with the patches. Internalization occurs 7of␣-factor, via thiopropionyl-Gly3 as a flexible, hydrophilic linker next, concomitant with actin assembly at patches. Additionally, (Fig. 1A). These labeled pheromones maintained biological activity, endocytic vesicles move toward early endosomes on actin cables. as assessed by induction of mating morphology in a cells, although Early endosomes also associate with actin cables, and they actively the activities were Ϸ25- to 50-fold less than for wild-type ␣-factor move toward endocytic sites to capture vesicles being released (see Fig. 6A, which is published as supporting information on the from the plasma membrane. Thus, endocytic vesicle formation and PNAS web site). Ste2p receptor-dependent binding and internal- capture of the newly released vesicles by early endosomes occur in ization of Alexa Fluor-594 (A594)-␣-factor indicated that A594-␣- a highly concerted manner, mediated by the actin cytoskeleton. factor is specifically internalized by receptor-mediated endocytosis (Fig. 1 B and C). When added to cells, A594-␣-factor (and actin ͉ cytoskeleton ͉ endocytosis ͉ endosome A488-␣-factor, data not shown) was first seen in internal endocytic compartments by 5 min (Fig. 1B; and see Movie 1, which is CELL BIOLOGY n recent years, live-cell imaging of endocytic events has proved published as supporting information on the PNAS web site). By 10 Iextremely powerful in yeast and other cell types for revealing min, A594-␣-factor began to concentrate in the vacuole and in mechanistic principles of endocytic internalization (1–7). In bud- bright structures that often were proximal to the vacuole. By 20 min, ding yeast, dynamics of at least two-dozen endocytic proteins have the ␣-factor was mostly in the vacuole (Fig. 1B; Movie 1). been analyzed by real-time analysis of GFP fusions, and effects of Recent studies showed that cortical actin patches are sites of numerous mutants on pathway dynamics have been quantitatively bulk-phase, clathrin-mediated endocytic internalization (2, 3, 5). analyzed. Although much is known about the dynamics and regu- Therefore, it is reasonable to ask whether receptor-mediated en- lation of the endocytic machinery as a result of these studies, a full docytosis of ␣-factor also occurs via these patches. To reveal the appreciation of the process depends on being able to analyze in real spatiotemporal relationships between cargo molecules and endo- time the transit of an endocytic cargo through the pathway. This cytic proteins, we tagged Abp1p, a marker for actin assembly at analysis is necessary so that functions such as cargo recruitment, endocytic sites, with monomeric red fluorescent protein (mRFP) concentration, internalization and trafficking can be attributed to and imaged the cells in real time as they endocytosed A488-␣- specific steps in the assembly and dynamics of the endocytic factor. Using total internal reflection fluorescence (TIRF) micros- machinery. copy, we observed A488-␣-factor as fluorescent spots moving An ideal cargo for such studies would be a fluorescent molecule diffusely on the cell surface (Fig. 1D Left; and see Movie 2, which that could be introduced to the cell externally, bound to cell-surface is published as supporting information on the PNAS web site). receptors, and then taken up by the endocytic machinery, such that Two-color analyses revealed that Abp1p (viewed by epifluores- the full history of the molecule would be known, and its fate could cence) joined preexisting A488-␣-factor spots (viewed by TIRF be followed as a function of time. Such a cargo molecule could be optics), and then both molecules disappeared concomitantly (Fig. used to define operationally the different compartments of the 1D; Movie 2). We found that, within the plane of the plasma endocytic pathway. The lipophilic dye FM4–64 and Ste2-GFP, an membrane, A488-␣-factor spots have a highly motile state and a integral membrane protein that is taken up by endocytosis, have nonmotile state. As shown in kymographs, all A488-␣-factor spots previously been used to label endocytic compartments fluores- in the nonmotile state are eventually joined by Abp1p and then cently in budding yeast. FM4–64 is introduced to cells externally internalized (100%; n ϭ 55) (Fig. 1E). In contrast, Abp1p never and it is a good marker for bulk-phase endocytosis (3, 5). However, once inside the cell, FM4–64 is transported along bifurcating pathways, with some dye entering a recycling pathway and the rest Conflict of interest statement: No conflicts declared. traveling to the vacuole (8); therefore, FM4–64’s utility for unam- Abbreviations: TIRF, total internal reflection fluorescence; LatA, Latrunculin A; mRFP, biguously labeling internal endocytic compartments to reveal spa- monomeric red fluorescent protein. tiotemporal features of the downstream pathway is limited. Ste2p is †J.Y.T. and J.T. contributed equally to this work. a receptor for the peptide ␣-factor, which is a mating pheromone. §To whom correspondence should be addressed. E-mail: [email protected]. Ste2-GFP has been used to mark endocytic compartments when © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601042103 PNAS ͉ April 11, 2006 ͉ vol. 103 ͉ no. 15 ͉ 5793–5798 Downloaded by guest on September 29, 2021 Fig. 1. Structure and localization of fluorophor-conjugated ␣-factor. (A) Diagram of Alexa Fluor-488 (A488)- and Alexa Fluor-594 (A594)-␣-factor. (B and C) Ste2p receptor-dependent binding and internalization of Alexa-␣-factor. Alexa-␣-factor was added to wild-type (B)orste2⌬ (C) cells and was followed through the endocytic pathway for the indicated times. (D, F, and G) A488-␣-factor (TIRF optics) appeared in endocytic patches before Abp1p and Sla1p but after Ede1p (epifluorescence). Shown are single frames from the GFP and the RFP channels of the movie and a merged image (Upper) and time series of single patches from wild-type cells expressing the indicated fluorophor-tagged proteins (Lower). The time to acquire one image pair was 2 s. (E) Kymographs of time-lapse images collected at 2-s intervals. Arrows in D mark where the kymograph was generated. Numbers and the direction of the arrows in D correspond to those in E. Arrowheads (E Right) indicate independent A488-␣-factor-labeled spots. (H) Localization of A594-␣-factor and Sla1-GFP in cells treated with 200 ␮M LatA. After incubating cells expressing Sla1-GFP with 200 ␮M LatA at 25°C for 30 min, cells were incubated with A594-␣-factor at 0°C for 30 min in minimal medium lacking glucose in the continued presence of 200 ␮M LatA. The images were acquired at 2 min and 20 min after washing out unbound Alexa-␣-factor with glucose-containing medium and warming cells to 25°C in the continued presence of 200 ␮M LatA. (Scale bars, 2.5 ␮m.) joined the highly motile A488-␣-factor spots (n ϭ 120) (Fig. 1E out its lifetime (n ϭ 100) (Fig. 6 B and C). This behavior is similar Right, arrowheads). Similar to Abp1p, the endocytic coat protein to what has been described for clathrin, although both clathrin and Sla1p (2, 3) also joined preexisting A488-␣-factor spots and was Sla1p persist after Ede1p disappears, and, in contrast to Ede1p, they cointernalized with them (100%; n ϭ 47) (Fig. 1F; and see Movie both are internalized (see ref. 3 and Fig. 6B). Interestingly, we 3, which is published as supporting information on the PNAS web observed that Ede1p forms patches that are subsequently joined by site). Because TIRF was used to image A488-␣-factor and epiflu- A488-␣-factor. Thus, endocytic sites form before ␣-factor recruit- orescence to image Sla1p, A488␣-factor occasionally disappeared ment (Fig. 1G; and see Movie 4, which is published as supporting before Sla1p. Our observations establish that cortical actin patches information on the PNAS web site), consistent with recent findings are sites of receptor-mediated ␣-factor internalization, an impor- in mammalian cells (7). The movement and disassembly of Sla1p tant conclusion, because Chang et al. (9) recently proposed that patches are known to be inhibited by Latrunculin A (LatA) ␣-factor may be internalized by a pathway independent from the treatment (2).