Using a limited number of erg
Δ strains, we have previously shown that specific sterol structures are necessary for receptor-mediated and fluid-phase marker endocytosis (Munn et al., 1999
). These studies did not tell us, however, whether sterol structures are required at multiple steps and whether the sterol structural requirements are the same for different processes. Therefore, we extended our endocytic analysis by using additional erg
Δ strains, each accumulating sterols with distinct structural differences to ergosterol and an additional endocytic marker that permits analysis of postinternalization steps. We provide evidence that there are multiple roles for sterols in yeast endocytosis. First, specific sterols are required in receptor-mediated endocytosis at or before receptor hyperphosphorylation arguing for a specific role at an early step in the process. Second, based on experiments analyzing FM4-64 and LY accumulation, sterol structures also have a role at a postinternalization step, which seems to be general. Third, some erg
Δ mutants show a fragmented vacuole. As discussed below, the sterol structural requirements for these processes are different.
In contrast to most known endocytic factors necessary for ligand-induced Ste2p-internalization, sterol structures are required before or at receptor modification. Based on the ligand competition studies, sterol structures seem to function subsequent to ligand-receptor interaction because changes in the sterol composition did not impair Ste2p function with regard to its ability to bind α-factor. Ste2p was also able to undergo a conformational change because exposure of α-factor induced the mating response (our unpublished observations). We cannot, however, exclude the possibility that aberrant sterols can support conformational changes leading to signaling, but not to receptor hyperphosphorylation.
Δ and erg3
Δ cells, whose aberrant sterols did not support α-factor internalization, Ste2p was not significantly hyperphosphorylated in response to binding of the pheromone. Hyperphosphorylation of serine/threonine residues in the cytoplasmic tail of Ste2p is a prerequisite for subsequent ubiquitination, the actual internalization signal (Hicke et al., 1998
; Shih et al., 2000
). The only kinases known to be involved in receptor phosphorylation are the redundant yeast casein kinase I homologs Yck1p and Yck2p. Similar to sterols, Yck proteins act early in receptor-mediated internalization because in yck-ts
cells, Ste2p is not internalized due to lack of hyperphosphorylation and ubiquitination after exposure to α-factor (Hicke et al., 1998
; Feng and Davis, 2000b
). Therefore, the endocytic internalization phenotypes of these erg
Δ mutants and yck-ts
cells (Friant et al., 2000
) are the same. They are defective in receptor-mediated endocytosis, due to a lack of receptor modification, but competent for the internalization step itself. One possible explanation for the lack of receptor phosphorylation in these erg
Δ mutants could be the inability to recruit the Yck kinases to their site of action at the plasma membrane. It is not yet known whether these kinases directly phosphorylate Ste2p.
Not all endocytic erg
Δ phenotypes can be explained by impairment of Yck kinase function because yck-ts
cells are not defective in fluid-phase endocytosis (Friant et al., 2000
). Changes in sterol composition affected both receptor-mediated and fluid-phase endocytosis in erg2
Δ, and erg3
Δ cells (this study) (Munn et al., 1999
). Importantly, the erg3
Δ and erg4
Δ cells displayed no defect in receptor-mediated endocytosis enabling us to separate the sterol requirement for receptor modification from a second requirement at a postinternalization step of endocytosis. More specifically, erg4
Δ cells were capable of internalizing FM4-64, but exhibited a delay in postinternalization movement of this membrane marker to vacuoles. It is noteworthy that the sterol structural requirement for fluid-phase endocytosis of a water-soluble dye may be different than that of a membrane-intercalating dye because erg3
Δ cells exhibited a strong defect in LY accumulation, but transport of FM4-64 was only slightly affected. The opposite results were seen for erg4
Δ cells. In agreement with a postinternalization defect, we observed accumulation of larger dot-like structures in erg3
Δ cells that contained Ste2p and were reminiscent of late endosomes (Figure ). These structures were not as conspicuous in WT cells. Based on FM4-64 and LY data, the sterol requirement for postinternalization is likely to affect all endocytic traffic.
To determine whether the sterol requirements for the endocytic processes are different, we extended our initial analysis (Munn et al., 1999
) in correlating the endocytic defects with the structural changes in the sterol molecule. As shown in Table , each erg
Δ strain accumulated a distinct set of sterols that differed from ergosterol in specific structural features. For clarity, the structures of the most abundant sterols (>10%) of each strain are shown with the observed endocytic phenotypes (Figure ). The predominant sterol in WT cells was ergosterol (Table ), a sterol containing two double bonds in the B-ring at C-5,6 and C-7,8, a double bond at C-22,23, and a methyl group (C-28) on C-24 in the side chain (Figure ). Based on the endocytic phenotypes and sterol analyses of erg2
Δ, and erg2
Δ, we suggested that the desaturation of the B-ring, but not the side-chain methylation at C-24,28, is critical for internalization of Ste2p (Munn et al., 1999
). More specifically, a single double bond at C-8,9 was not sufficient to support receptor internalization, whereas two double bonds, at C-5,6 and C-7,8 or C-8,9, allowed internalization. If the previous suggestions were true then erg2
Δ cells, which accumulated only sterols with a single C-8,9 desaturation, all of which contained a methyl or methenyl group on C-24, would be expected to exhibit a strong block in receptor-mediated internalization as reported for erg2
Δ cells (Munn et al., 1999
). However, erg2
Δ cells exhibited only partially reduced α-factor internalization. These results suggest that B-ring desaturation is not the sole structural requirement for its internalization. Mutant erg3
Δ cells, which accumulated a mixture of sterols with a single C-7,8 or C-8,9 desaturation lacking methylation on C-24, showed a severe block in α-factor uptake similar to that of erg2
Δ cells (Munn et al., 1999
). Thus, a single desaturation at C-7,8 may not be sufficient to drive receptor-mediated internalization in the absence of methylation on C-24.
Figure 9 Comparison of endocytic phenotypes and sterol structures of ergΔ cells. The chemical structures of the most abundant sterols (>10%; as determined in Table ) were grouped with the different endocytic and vacuolar (more ...)
Based on the ability to take up α-factor with WT rates in erg3
Δ and erg4
Δ cells, changes in the sterol composition did not necessarily lead to a Ste2p-internalization defect. Mutant erg3
Δ cells, containing sterols with a single desaturation at C-7,8 with proper side-chain methylation were able to support internalization as well as ergosterol. No desaturation in the side-chain (i.e., lack of C-22,23 desaturation) or a single desaturation at C-24,28 (erg4
Δ) instead of at C-22,23 have no or little effect on internalization (also see erg2
Δ sterols). Although we cannot exclude a role of minor sterols, we conclude that contrary to our previous interpretation (Munn et al., 1999
), a combination of both proper B-ring desaturation and side-chain methylation is most likely required for efficient ligand-induced Ste2p-internalization. Therefore, the overall structure of the ergosterol molecule seems to be important for this endocytic step.
Interestingly, all cells we analyzed with the erg6
Δ mutation showed an increase in peripheral staining of FM4–64 after the dye had been internalized. Although the exact nature of this peripheral staining is not known yet, this suggests that the side chain methylation performed by Erg6p may play an important role(s) in the endocytic pathway. If the increased peripheral staining were due to increased recycling, this would be similar to what has been found in mammalian cells. Internalized GPI-anchored proteins are recycled back to the cell surface more rapidly when cells are depleted of cholesterol (Mayor et al., 1998
In contrast to receptor-mediated internalization, the structural sterol requirements for FM4-64 internalization were more difficult to define because all ergΔ strains internalized the dye well. As particularly evident when comparing the endocytic phenotypes for erg4Δerg5Δ cells (Figure ), the structural requirements for internalization and postinternalization steps of fluid-phase endocytosis were different. A change in a single desaturation of the side chain from C-22,23 (present in ergosterol) to C-24,28 (present in ergosta-5,7,24-trienol, the most abundant sterol in erg4Δerg5Δ cells), led to a strong delay in postinternalization movement of FM4-64 to vacuoles. In contrast, these structural changes had no effect on Ste2p-internalization. The explanation for the role(s) of sterol in postinternalization will obviously be complex and require further analysis.
We also noticed that erg
Δ cells frequently had fragmented vacuoles consistent with a role of ergosterol in homotypic vacuole fusion (Kato and Wickner, 2001
). The ergosterol requirement for vacuole integrity was distinct from the one for receptor modification because the erg3
Δ mutant showed one of the most severe receptor modification defects, but had normal vacuoles, whereas the erg3
Δ mutant showed no receptor modification phenotype, but had strongly fragmented vacuoles (Figures and ). Vacuolar morphology could also indicate a loss of some vacuolar function, for example vacuole acidification. To obtain a crude measure of vacuole acidification we measured quinacrine accumulation in the vacuoles of the ergΔ
mutant strains. All of the ergΔ
mutants were able to accumulate quinacrine in their vacuoles, suggesting that there are no major defects in vacuole acidification due to the ergΔ
mutations (our unpublished observations).
The ability of erg
Δ cells, even of those displaying a strong defect in Ste2p internalization, to take up the FM4-64 is in agreement with the fact that their actin cytoskeleton organization was not obviously affected as judged by staining of F-actin. This endocytic behavior differentiates the erg
Δ strains from other known end
mutants involved in building or regulating the actin cytoskeleton because these end
mutants are defective in internalization of FM4-64 (D'Hondt et al., 2000
). It is therefore unlikely that in yeast, sterols provide a direct or indirect attachment site for the actin cytoskeleton. Consistent with these results, no significant difference in the extractability of Rvs167p, End3p, and End4p, proteins that interact with the actin cytoskeleton and that are required for Ste2p internalization (Geli and Riezman, 1998
; D'Hondt et al., 2000
) was observed when comparing erg
Δ mutant and WT cell extracts (our unpublished observations).
It is possible that the endocytic defects in erg
Δ mutant cells may be due to the inability of the aberrant sterols to associate with sphingolipids and thus to organize the lipid environment of the membrane (e.g., “lipid rafts”). We were unable, however, to establish a correlation between detergent solubility of Gas1p, a plasma membrane marker of yeast lipid rafts (Bagnat et al., 2000
), and the ability of cells to internalize α-factor. On the other hand, the structural determinants we have identified as important for sterol function in endocytosis are similar to the structural determinants shown to influence sphingolipid/sterol domain formation in model membranes (Xu et al., 2001
). These apparently conflicting data could be explained by the association of sterols with different types of lipid rafts or microdomains that coexist in membranes or vary between membranes and exhibit distinct physical properties. If so, it may also provide an explanation for the presence of the different sterol requirements in receptor-mediated and fluid-phase internalization as well as in postinternalization. Alternatively, the erg
mutations could cause an alteration in the sterol concentration in the plasma membrane, and the different assays, Gas1p solubility and endocytosis, may be affected by different threshold concentrations of sterols. Another possibility is that the aberrant sterol structures affect the properties of whole bilayers and that the different structural variants of sterols produced in the mutant strains affect the bilayer structures in different ways.
It is also possible that sterols may function in yeast internalization independently of their association with sphingolipids. It should be noted that although suggested (Zanolari et al., 2000
), a direct role of sphingolipids in yeast endocytosis has not been demonstrated yet. Clearly, the role of ergosterol differs from that of sphingoid bases, which are sphingolipid precursors recently shown to be required for endocytic internalization (Zanolari et al., 2000
). Hyperphosphorylation of Ste2p is not impaired in lcb1–100
cells defective in sphingoid base synthesis and sphingoid bases are necessary for proper actin cytoskeleton morphology (Zanolari et al., 2000
Our study provides evidence that ergosterol functions differently in internalization than other previously described components of the yeast endocytic machinery, with the exception of yeast casein kinase I and Akr1p, which is required to recruit casein kinase I to the membrane (Feng and Davis, 2000a
). Although many of the other known endocytic factors, including sphingoid bases, act through the actin requirement, ergosterol is important early in Ste2p internalization and sterol function seems to be independent of actin. In addition to receptor hyperphosphorylation, ergosterol is required for postinternalization process(es) in endocytosis, and the availability of a variety of erg
Δ mutants displaying different endocytic phenotypes will allow us to investigate the roles of sterols in these processes in more detail. It is clear from our data that distinct structural properties of the sterol molecule will be important for its role in the various steps of endocytosis.