Correspondence to: Nicholas G. Davis, Departments of Surgery and Pharmacology, Wayne State University School of Medicine, Elliman Building, Room 1205, 421 E. Canfield, Detroit, MI 48201. Tel:(313) 577-7807 Fax:(313) 577-7642
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Abstract |
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The yeast a-factor receptor (Ste3p) is subject to two mechanistically distinct modes of endocytosis: a constitutive, ligand-independent pathway and a ligand-dependent uptake pathway. Whereas the constitutive pathway leads to degradation of the receptor in the vacuole, the present work finds that receptor internalized via the ligand-dependent pathway recycles. With the a-factor ligand continuously present in the culture medium, trafficking of the receptor achieves an equilibrium in which continuing uptake to endosomal compartments is balanced by its recycling return to the plasma membrane. Withdrawal of ligand from the medium leads to a net return of the internalized receptor back to the plasma membrane. Although recycling is demonstrated for receptors that lack the signal for constitutive endocytosis, evidence is provided indicating a participation of recycling in wild-type Ste3p trafficking as well: a-factor treatment both slows wild-type receptor turnover and results in receptor redistribution to intracellular endosomal compartments. Apparently, a-factor acts as a switch, diverting receptor from vacuole-directed endocytosis and degradation, to recycling. A model is presented for how the two Ste3p endocytic modes may collaborate to generate the polarized receptor distribution characteristic of mating cells.
Key Words: endocytosis, Saccharomyces cerevisiae, pheromones, cell surface, endosomes
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Introduction |
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Recycling of cell surface receptors in mammalian cells allows the receptor protein to participate in multiple rounds of ligand binding and internalization (
In the yeast Saccharomyces cerevisiae, a number of plasma membrane proteins have been found to undergo a ubiquitin-dependent endocytosis that delivers the internalized protein to the vacuole (the yeast lysosome) for degradation (
The a-factor receptor (Ste3p), one of the two G proteincoupled receptors directing sexual conjugation in yeast, is subject to two distinct modes of endocytosis: a ligand-independent, constitutive uptake mode as well as a ligand-dependent mode. To date, the Ste3p constitutive uptake mode has been the more intensively studied of the two (
To date, the ligand-dependent uptake of Ste3p has been followed only under conditions where rapid constitutive endocytosis is impaired (
The present work that concentrates on Ste3p ligand-dependent uptake finds that the two Ste3p uptake modes also differ in terms of the fate of the internalized receptor. Rather than the vacuolar degradation associated with constitutive endocytosis, ligand-dependent uptake links instead to recycling: receptor internalized via this pathway to endosomal compartments recycles back to the cell surface.
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Materials and Methods |
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Strains
The strains used in this work are listed in Table 1. New strains, all isogenic to NDY341 (
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Cell Culture, Pheromone Treatment, Protease Shaving, Protein Extract Preparation, and Western Analysis
A 90-min period of receptor expression from GAL1-driven receptor constructs was initiated from log-phase cultures growing in YPR medium (YP medium [1% yeast extract, 2% peptone] with 2% raffinose) with the addition of galactose to 2%, and was terminated with the addition of glucose to 3%.Pheromone was added 30 min after the addition of glucose or together with the glucose in the experiments that used wild-type Ste3p (see Fig 5). Pheromone addition involved adding 0.5 vol of the a-factorcontaining culture supernatant from EG123 cells carrying pKK16 (2µ STE6, MFA2; 365p (see Fig 1 C), extracts were prepared from the culture aliquots as previously described (
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Quantitative Methods
Loss of Ste3 antigen to turnover or to protease shaving was quantitated using film densitometry. Films of Western blots developed with enhanced chemiluminescent reagents (Amersham Pharmacia Biotech) were digitally scanned and the resulting TIFF files quantitated with NIH Image software. Measurements entailed comparison of the Ste3 antigen that survived turnover or protease digestion to a standard curve generated by dilution of the initial sample (i.e., receptor present at the 0-h timepoint of a turnover experiment or in the case of protease-shaving experiments, receptor present in the control sample, untreated with proteases).
Recycling Experiments
Recycling was followed with two different protocols. In the first protocol (used for Fig 3), after a 45-min a-factor treatment (leading to the internalization of >50% of the 365 receptor protein), 6 x 108 cells were collected twice by centrifugation, washed with fresh 30°C YPD medium (YP medium with 2% glucose), and restored to culture. As a control for the effects of the a-factorremoving "wash" steps, half of the cells in parallel, were washed and restored to culture using fresh a-factorcontaining medium (YPD plus an additional 0.5 vol of the a-factorcontaining supernatant; see above). At various times after restoring the cells to culture, aliquots were removed and receptor distribution was monitored via the protease-shaving protocol (see above).
For the second protocol, the 45-min a-factor treatment was followed directly by protease shaving to destroy that portion of the receptor population that remained at the cell surface after the pheromone treatment. This preparative Pronase treatment was a scaled-up version of the analytical protease-shaving protocol (see above): 6 x 108 a-factortreated cells were digested with 2,000 units of Pronase in a 9-ml vol. After this shaving, cells were collected by centrifugation and restored to culture in 25 ml of 30°C YPDS medium (YPD medium with 1 M sorbitol). Then the cell surface return of the surviving, intracellular receptor population was followed using the analytical protease-shaving protocol as described above.
Indirect Immunofluorescence
NDY1181 cells expressing a 3xHA epitope-tagged version of Ste3365p (epitope tags fused to receptor COOH terminus) were cultured and treated with a-factor as described above. Cells were fixed, spheroplasted, and treated for indirect immunofluorescence as previously described (
365(3xHA)p used a 1:1,000 dilution of the HA.11 mAb (Berkeley Antibody Co.) followed by a Cy3-conjugated donkey antimouse IgG secondary antibody used at a 1:500 dilution. For detection of the vacuolar membrane protein alkaline phosphatase (ALP), an affinity-purified, rabbit polyclonal anti-ALP (provided by Steve Nothwehr, University of Missouri, St. Louis, MO) that had been pre-absorbed against pho8
cells (ALP is encoded by PHO8) was used at a dilution of 1:6. This was followed by a 1:500 dilution of biotinylated goat antirabbit IgG secondary antibody, and then fluorescein-conjugated strepavidin at 1:500. All secondary antibodies, as well as the fluorescein-conjugated strepavidin, were from Jackson ImmunoResearch Laboratories. Images were captured using a Nikon Eclipse 600 (Nikon) equipped with a Princeton Instruments Micromax CCD camera (Roper Scientific Princeton Instruments Inc.).
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Results and Discussion |
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Kinetics of Ligand-induced Endocytosis and Turnover
To focus on the Ste3p ligand-dependent uptake mode, we have made use of a receptor mutant fully defective for the constitutive mode: Ste3365p lacks the COOH-terminal 105 residues of the receptor CTD including the COOH-terminal PEST-like constitutive endocytosis signal (Fig 1 A). Consequently, Ste3
365p stably accumulates at the plasma membrane. With a-factor treatment, Ste3
365p is internalized and delivered ultimately to the vacuole where it is degraded (
365p-expressing cells with a-factor. After a pulse of receptor synthesis from the GAL1 promoter, a-factor is added, and receptor internalization is monitored using a protease-shaving protocol in which intact cells are treated with proteases (Fig 1 B). Receptor localized to the cell surface is susceptible to digestion by the added extracellular proteases, while receptor that localizes to intracellular compartments (e.g., endosomes or vacuole) is protected from digestion (
prb1
to avoid loss of receptor protein to the normal vacuolar turnover mechanism during the experimental timecourse.
Before a-factor addition, >90% of the receptor protein was found to be susceptible to digestion by the added proteases (Fig 1 B, 0 min timepoint), indicating that receptor initially is localized at the cell surface. In the first 40 min after a-factor addition, receptor becomes increasingly resistant to digestion, consistent with an increasing fraction of the receptor population being internalized. During this initial phase of the timecourse, we estimate a t1/2 for internalization of 35 min (Fig 1 D). Subsequently, the rate of receptor internalization appears to slow, reaching a plateau in which 55 to 65% of the receptors are internalized and 35 to 45% remain exposed at the cell surface.
In a parallel experiment, the rate of a-factorinduced Ste3365p turnover was assessed in wild-type PEP4+ PRB1+ cells (Fig 1 C). Cells treated with a-factor show a slow loss of receptor protein over the 2-h time course. Unlike the constitutive endocytosis of wild-type Ste3p where receptor uptake and turnover are tightly coupled (
365p indicates that turnover lags substantially behind internalization (Fig 1 D). Whereas 50% of the receptor is internalized over the initial 35 min of a-factor treatment, it takes 80 min for 50% of the receptor protein to be degraded. Thus, before its degradation, a relatively large fraction of the receptor shows a surprisingly stable accumulation inside the cell.
Internalized Receptor Accumulates in an Endosomal Compartment
To identify the intracellular sites of receptor accumulation, we have used indirect immunofluorescence microscopy to follow the a-factorinduced internalization of an HA epitope-tagged 365 receptor (Fig 2). Previous analysis that demonstrated the vacuole as the final destination for the a-factorinduced uptake of Ste3
365p, used both a pep4
strain background and a long period of pheromone treatment (90 min) (
Recycling
The biphasic kinetics observed for 365 internalization (Fig 1 D) might be explained by ongoing receptor recycling. The plateau seen for receptor internalization 40 min after a-factor addition (Fig 1 D) could be indicative of an equilibrium in which ongoing internalization is balanced by the recycling return of receptor back to the cell surface. Drawing from examples of recycling in mammalian cells, one might anticipate that after internalization to an endosomal compartment, ligand and receptor may dissociate with unliganded receptor returning to the cell surface to repeat the endocytic cycle (rebinding ligand and re-internalizing). Such a cycle of uptake and return could be interrupted simply through removal of the uptake stimulus, i.e., the ligand. Removal of the ligand from the culture medium should block further uptake, allowing the potential recycling return of the receptor to the cell surface to be visualized. Such an experiment is shown in Fig 3. After a 45-min a-factor treatment of Ste3
365p-expressing cells that resulted in
50% of the receptor protein being internalized (Fig 3), cells were removed from culture and restored either to fresh medium lacking a-factor or as a control, to medium in which a-factor is maintained at its original concentration. When restored to medium containing a-factor, we find that an intracellular pool of receptor protein is maintained, with
50% of the receptor protein resisting digestion by added proteases (Fig 3). As the cells used for this experiment are PEP4+, a slow, a-factorinduced, receptor turnover also is apparent. Quite a different result is obtained when a-factor is removed from the culture medium. With withdrawal, there is a clear, time-dependent redistribution of the receptor back to the cell surface: 30 and 60 min after withdrawal, we find that 80 and 90% of the total receptor protein, respectively, is surface localized.
To focus more directly on the endosome-to-surface transport component of recycling, we have developed an alternative experimental approach where recycling is followed from a condition that has all the receptor protein initially localized endosomally (Fig 4 A). To establish this initial condition, we use the protease-shaving technique to eliminate the portion of the receptor population that remains at the cell surface subsequent to a 45-min a-factor treatment period. Cellular energy metabolism is poisoned during the protease treatment step to block the possibility of continued membrane trafficking. After proteolysis, energy poisons are removed, the shaved cells are restored to culture, and the changing receptor localization is monitored. A clear time-dependent return of internalized receptor to the cell surface is seen (Fig 4 A). This recycling is energy dependent as it is blocked for a control culture where the presence of the energy poisons is maintained (Fig 4 A).
The experiments above define a recycling pathway associated with the ligand-dependent endocytosis of the 365 Ste3p CTD tail truncation mutant. In addition to the PEST-like signal, the 105-residue
365 deletion interval also could include sequences that participate in other, unforeseen aspects of receptor trafficking. For instance, it is possible that endosomal accumulation of internalized Ste3
365p could reflect the loss of hypothetical receptor sequences that specify endosome to vacuole transport. More subtle receptor mutations that disable constitutive endocytosis have been generated in recent studies of the Ste3p PEST-like signal (
R mutation is a Lys-to-Arg substitution of the three lysine residues of the PEST-like signal that serve as the redundant ubiquitin acceptor sites (Fig 1 A). The triply substituted Ste3(3K
R)p fails to be ubiquitinated, fails to undergo constitutive uptake, and consequently accumulates at the cell surface (
R)p does remain competent for ligand-dependent endocytosis, with kinetics of uptake similar to that observed for the
365 truncation mutant (
R reveals a turnover lag like that seen for Ste3
365p (Fig 1 D): a-factorinduced internalization of Ste3(3K
R)p is relatively rapid whereas the associated turnover lags slowly behind (data not shown). To test if Ste3(3K
R)p also undergoes recycling, we have applied the protocol used for Ste3
365p in Fig 4 A. Again, we see much the same result as was obtained for
365: internalized 3K
R receptor shows a time- and energy-dependent return to the cell surface (Fig 4 B).
Recycling of the Wild-type Receptor
How might the ligand-induced receptor recycling mechanism impact wild-type Ste3p? In the absence of ligand, Ste3p is subject to a rapid degradative endocytosis. As we have seen above (Fig 3 and Fig 4), the ligand-dependent endocytosis of 365 and 3K
R mutant receptors links primarily to recycling. If a-factor provides the switch that converts wild-type Ste3p uptake from the degradative to the recycling mode then we should expect several changes after treatment of wild-type Ste3p-expressing cells with a-factor. First, considering the pronounced intracellular accumulation of Ste3
365p after a-factor treatment (Fig 1 and Fig 2), we might expect a ligand-induced redistribution of the wild-type receptor, with an increased proportion of the receptor population localizing intracellularly to endosomal compartments. Second, with recycling induced instead of degradation, a-factor treatment might also lead to an overall slowing of the turnover rate.
In Fig 5 A, we have examined the effects of a-factor treatment on the localization and turnover of wild-type Ste3p. Surface localization of the receptor is again assessed using the protease-shaving protocol. Unlike the 365 and 3K
R receptors, the residency of wild-type Ste3p at the plasma membrane is short-lived (5 to 10 min for cells growing at 30°C). To maximize exposure of the receptor to ligand, a-factor was added together with glucose at the end of a 90-min period of galactose-induced receptor synthesis. At that time (Fig 5 A, 0 min timepoint),
60% is surface localized and
40% is localized intracellularly. The bulk of the intracellular receptor population is newly synthesized receptor that has not yet arrived at the cell surface: consistent with this, the proportion of receptor that is surface localized increases 30 min after the glucose-mediated shut-off of new receptor synthesis (Fig 5 A, 30 min, no a-factor timepoint).
Comparing receptor turnover and distribution in the a-factor treated cultures to that of the unstimulated control, we do indeed find effects consistent with those forecast for the induction of a recycling mode of endocytosis (Fig 5 A). 30 and 60 min after the a-factor challenge, a much higher fraction of the receptor population is found to distribute intracellularly compared with the mock-treated control. In addition, a subtle, yet reproducible slowing in the rate of receptor turnover for the a-factortreated cultures is also seen. A more substantial receptor stabilization may require exposure to high concentrations of a-factor as is thought to occur during the late stages of mating (
Pheromone treatment induces an array of responses including induced transcription, arrest of the cell cycle in G1, and a polarization of cell growth that results in a deformation of the cell body into a mating projection or "shmoo". Therefore, we have considered the possibility that the a-factor effects on Ste3p turnover and distribution might be indirect, resulting from interference by one of these other pheromone-induced responses. To this end, we have tested the consequence of deletion of the gene encoding the Gß subunit of the heterotrimeric ß
G protein (i.e., ste4
cells) on the a-factor effects on Ste3p turnover and redistribution. Activation of the heterotrimeric G protein is the first intracellular signaling step for the pheromone signaling pathway and ste4
cells fail to mount a pheromone response. Interestingly, ligand-induced endocytosis for both the a- and
-factor receptors is distinguished from the other pheromone responses by G protein independence: uptake of both proceeds in ste4
cells (
cells, indicates a lack of requirement for G proteininduced signaling (Fig 5 B). Thus, the noted effects of a-factor on Ste3p trafficking share the unique feature of ligand-dependent endocytosis: they too are G proteinindependent, indicating that they likely do result from a ligand-induced switch from a degradative endocytosis to a recycling mode.
Endocytic Recycling in Yeast
Our results with Ste3p recycling fit nicely within recycling paradigms established for a variety of mammalian receptors. Ligand binding triggers uptake of surface receptor to an endosomal compartment. By analogy to the mammalian paradigm, we expect at this point, dissociation of ligand from the receptor. Ligand may travel onward to the vacuole for degradation while the receptor returns to the surface for reutilization.
Endocytic recycling also has been suggested as participating in the trafficking of two other yeast membrane proteins, the chitin synthetase Chs3p and the vesicle-associated membrane protein (VAMP)-like vesicle soluble NSF attachment protein receptor (v-SNARE) Snc1p (-factor pheromone receptor (Ste2p) trafficking (
Endocytosis and Mating
What role might the two Ste3p endocytic modes play in the mating process? Recycling allows reutilization of the receptor. Thus, a-factor, in addition to inducing the synthesis of new receptor protein through induced STE3 transcription, also conserves preexisting receptor through recycling. In keeping with this, Ste3p levels are found to be dramatically elevated in mating MAT cells (Roth, A., and N. Davis, unpublished results). Recycling may also play a role in the redistribution of surface receptor that occurs during mating. During the late stages of mating, pheromone receptors concentrate within the polarized mating projections of their respective cells (
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Footnotes |
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1 Abbreviations used in this paper: ALP, alkaline phosphatase; CTD, COOH-terminal cytoplasmic tail domain.
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Acknowledgements |
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We thank Steve Nothwehr for affinity-purified anti-ALP antibodies and Jeff Loeb for the use of his microscope and help with the immunofluorescence microscopy. We thank Sandy Lemmon and Bob Fuller for helpful suggestions. We thank Amy Roth for her early work on the ligand-dependent Ste3p endocytosis and for helpful comments on this manuscript.
This work was supported by a grant from the National Science Foundation (MCB 99-83688).
Submitted: 11 August 2000
Revised: 19 September 2000
Accepted: 19 September 2000
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References |
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