Here we describe a comprehensive genome wide screen for genes required for the proper intracellular sorting of Gap1p in response to nitrogen source. Most of the mutations identified that increase the proportion of Gap1p sorted to the plasma membrane are in class E VPS genes that have been shown to be required for the formation of the MVE. These results establish that when Gap1p cannot enter the inwardly budding vesicles of the MVE efficient recycling of Gap1p from the endosome to the plasma membrane is possible. Genetic and physiological tests of the conditions under which cycling of Gap1p from endosome to the plasma membrane occurs show that high concentrations of amino acids can block cycling. Regulation of this cycling step appears to be the major physiological input of intracellular amino acid concentration in the overall process of regulating the amount of Gap1p delivered to the plasma membrane. The cycling step is also under genetic control by a collection of genes including LST4 and LST7, and these genes are potential targets for regulation by amino acids.
A second stage of the intracellular sorting itinerary of Gap1p with the potential to be regulated by intracellular amino acids is ubiquitination of Gap1p, which enables Gap1p to be targeted to the endosome in the first place. The ubiquitination state of Gap1p is set by both ubiquitinating and deubiquitinating processes, and we developed an assay for the effect of intracellular amino concentration on the accumulation of ubiquitinated Gap1p based on the finding that deubiquitination of Gap1p could largely be eliminated by deletion of DOA4. In a doa4Δ genetic background a large fraction of the total Gap1p is in a polyubiquinated state, but the fraction of Gap1p that is ubiquitinated does not change significantly in the presence or absence of amino acids. Moreover, mutations such as lst4Δ, which have an effect on Gap1p sorting similar to growth in high amino acid concentrations, also does not give rise to a significant increase in the fraction of Gap1p that is polyubiquitinated. These results lead us to conclude that poyubiquitination of Gap1p alone does not suffice to explain the dramatic effect that amino acids exert in the down-regulation of Gap1p. Regulation of Gap1p recycling must constitute an additional and very likely more important target for the regulation of Gap1p sorting by amino acid levels.
outlines the two proposed decision points in Gap1p sorting in the late secretory pathway, showing the steps affected by intracellular amino acids and each of the different classes of mutants known to affect the distribution of Gap1p in the cell. The first decision appears to take place in the trans
-Golgi and depends on Gap1p ubiquitination by the Rsp5p/Bul1p/Bul2p ubiquitin ligase complex (Helliwell et al., 2001
; Soetens et al., 2001
). The second sorting decision is made in the MVE, and at this stage Gap1p can either be recycled to the plasma membrane by an LST4
-dependent process or can be sorted into endosomal vesicles by ESCRT complex proteins.
Figure 13. Proposed model for regulated Gap1p sorting in the endosome. Newly synthesized Gap1p has two possible fates once it reaches the trans-Golgi: sorting to the plasma membrane where it is active for amino acid uptake or to the vacuole for degradation. Polyubiquitination (more ...)
We cannot, nor do we wish to, rule out the possibility that Gap1p recycling to the plasma membrane occurs by a direct MVE to plasma membrane transport step, as has been indicated for trafficking of misfolded Pma1p (Luo and Chang, 2000
). However, when the partitioning probabilities for sorting decisions in both the Golgi and MVE are considered, we believe the present data indicates that Gap1p normally cycles between the TGN and MVE compartments. In the presence of high intracellular amino acid concentrations (or in an lst4Δ
mutant) the amount of Gap1p delivered to the plasma membrane is only a few percent of the amount delivered in an ubiquitination defective mutant such as bul1Δ bul2Δ
. This result shows that in a wild-type cell most Gap1p is initially transported to the MVE compartment where the amino acid–dependent sorting decision takes place. In the absence of high intracellular amino acids (or in a subset of ESCRT mutants) Gap1p is efficiently cycled from the MVE to the plasma membrane. Importantly, when either Bul1p or Bul2p is overproduced, most of Gap1p can be returned to the VPS pathway, showing that Gap1p that is recycled from the MVE enters a compartment that can still be influenced by the rate of Bul-dependent ubiquitination. This Bul-dependent compartment is most likely the TGN and not the plasma membrane because endocytosis defective mutants have little effect on the amount of Gap1p partitioned to the plasma membrane. It appears that Gap1p may cycle between the TGN and MVE multiple times—the sorting probabilities in each of these compartments thus determining the overall partitioning of Gap1p between the plasma membrane and vacuole.
In agreement with our observations, Bugnicourt et al. (2004)
have recently shown that ESCRT mutants cause recycling of the uracil permease, Fur4p, to the cell surface. In this case, they observed a block in Fur4p recycling in double mutants simultaneously affecting components of the ESCRT and HOPS complexes (the latter is involved in vacuolar fusion events; see Wickner, 2002
). These observations were interpreted to indicate that Fur4p recycling is mediated by the HOPS-dependent pathway, which bypasses the Golgi. Similarly, Nikko et al. (2003)
observed that in S. cerevisiae
cells of the Σ1278b genetic background the cell surface accumulation of Gap1p in a bro1Δ
mutant in the presence of ammonia is abolished by a simultaneous mutation in VAM3
genes, also suggesting the possible existence of a recycling pathway from the vacuolar and/or late endosomal membranes to the cell surface independent from the Golgi. Although we do not rule out that such a pathway may also exist for Gap1p, in our genetic background (S288C) we found that vam3Δ
mutations have no significant effect on Gap1p sorting (as shown in and , and ). We find that null mutations in genes encoding for components of the HOPS complex (such as vps16Δ
caused significantly elevated levels of Gap1p activity rather than decreased levels that would be predicted if the HOPS complex were required for cycling of Gap1p to the plasma membrane. Moreover, these mutations did not have a significant effect on cycling of Gap1p to the plasma membrane in the presence of the class E vps mutation vps4Δ
. These results indicate that mutations in the HOPS complex may have a similar effect on Gap1p trafficking as ESCRT mutants that block progression of Gap1p from the MVE to the vacuole.
In contrast, mutations such as lst4Δ and lst7Δ, which block Gap1p recycling to the plasma membrane, cause a dramatic reduction in Gap1p activity in cells grown on medium without amino acids. This simple assay has allowed us to test other mutants known to participate in endosome to Golgi trafficking for a specific effect on Gap1p recycling. As shown in and and in , we did not detect a significant effect on Gap1p activity by mutations in the retrograde complex or in retromer components. These mutations did not interfere with the efficient cycling of Gap1p to the plasma membrane in the presence of an ESCRT mutation such as vps4Δ.
Interestingly, although mutations such as lst4Δ and lst7Δ have a profound effect on Gap1p activity, this and other mutations identified in the screen that block Gap1p cycling to the cell surface do not seem to cause a more generalized defect on trafficking as shown by the normal CPY sorting and FM4-64 recycling patterns in the corresponding null mutant strains (; and Gao and Kaiser, unpublished results). Taken together, the specificity of these different classes of cycling mutants for different cargo proteins implies the existence of at least two genetically distinct pathways for endosome-to-Golgi trafficking.
A recent report revealed a possible role of GGA proteins in facilitating transport of Gap1p from the TGN to a prevacuolar compartment (Scott et al., 2004
). Although a possible role of these proteins in increasing the efficiency of Gap1p vacuolar sorting rate may exist, Gap1p sorting to the VPS pathway does not absolutely depend on these functions because an lst4Δ gga1Δ gga2Δ
double mutant still shows constitutive sorting of Gap1p to the vacuole. Data shown by Scott et al. (2004)
indicated that the defect in vacuolar sorting observed in a gga1Δ gga2Δ
double mutant is only partial and that it may be related to defective endocytic trafficking. Because here we show that LST4
must have a role in Gap1p sorting that is independent from endocytosis of the permease, it is therefore possible that GGA-dependent sorting of Gap1p occurs only in specific steps of the vacuolar sorting of Gap1p (e.g., in the formation of endocytic vesicles).
Several recent studies have indicated a direct action of Rsp5p on different cargo at the MVE (Blondel et al., 2004
; Dunn et al., 2004
; Katzmann et al., 2004
; Morvan et al., 2004
). In all of these cases the corresponding cargo proteins accumulate at the MVE in an rsp5
-deficient background. By contrast, mutations affecting the polyubiquitinating machinery Rsp5p/Bul1p/Bul2p cause Gap1p to be localized at the cell surface, not the MVE. Most importantly in a bul1Δ bul2Δ
double mutant either the presence of high intracellular amino acids or a lst4Δ
mutation have no effect on the trafficking of Gap1p to the cell surface, indicating that in the absence of ubiquitination Gap1p never reaches the sorting step controlled by amino acids and Lst4p. The most straightforward explanation for this result is that amino acids and Lst4p control sorting at the membrane of the MVE and that the sorting step governed by Rsp5/Bul1/Bul2p action on Gap1p takes place at an earlier stage of the pathway, most likely in the trans
Although sharing similar features in their vacuolar sorting through the MVE pathway, other permeases have already shown differences with Gap1p in the particular steps governing their ubiquitination. For instance, although Rsp5p plays an important role in Fur4p MVE sorting, the polyubiquitination of the uracil permease does not seem to depend on Bul proteins (Blondel et al., 2004
The availability of Gap1p for its own recycling out of the MVE may be the result of a balance between the state of Gap1p polyubiquitination, its consequent recognition by ESCRT proteins, and the state activity status of both Lst4p and the components of the recycling sorting machinery for Gap1p in response to amino acid concentrations. Such regulation could determine the ability of any putative recycling machinery to compete for Gap1p interaction, with certain ESCRT machinery components specialized for recognition of Gap1p as MVE cargo. In this regard, our results show that different subsets of ESCRT proteins may have a more important role than others in the ability to retain Gap1p for its MVE sorting. Our results provide additional evidence that ESCRT proteins have differential roles in the sorting of specific MVE cargo, as suggested by previous works (Kranz et al., 2001
; Köhler, 2003
In agreement with previous observations made by Nikko et al. (2003)
in our experimental conditions a bro1Δ
mutant showed higher Gap1p activity than the rest of ESCRT mutations, reaching levels comparable to those displayed by mutations in the polyubiquitinating machinery or DOA4
. Although the combination with an lst4Δ
mutation rescued the ability of a bro1Δ
mutant to grow in the presence of low levels of ADCB by reducing Gap1p activity, this double mutant exhibited an unusually high percentage of Gap1p-GFP located at the cell surface. The similarity of doa4Δ
mutations on their effect on Gap1p sorting can now be explained by the recent discovery that Bro1p functions to recruitment of Doa4p to endosomal membranes (Luhtala and Odorizzi, 2004
Future analysis involving further characterization of the amino acids ability to modulate the activities of the steps controlled by Rsp5/Bul1/Bul2p, ESCRT proteins, Lst4p, Lst7p, and recycling machinery will help to elucidate how metabolic signals can change the final fate of Gap1p and other plasma membrane proteins.