The structure of the Vps15 WD domain is presented in . The protein folds as a seven-bladed propeller as predicted in a prior homology modeling analysis (5
). As described previously, the WD repeat units are not equivalent to the blades of the propeller, but are comprised rather of the fourth strand of one blade and the first three strands of the blade that follows (36
). The overall architecture of the Vps15 WD domain is similar to that of the transducin G protein β subunit (Gβ1
), and superposition of the structures with the CCP4 topological comparison program TOP yields a r.m.s. deviation of 2.7 Å2
for the Cα carbons (37
). There are several key differences in the overall structures of the two proteins. The N-terminus of Gβ folds as an α-helix that extends away from the propeller. In the case of Vps15, the corresponding region is largely unstructured and it packs against the bottom surface of the propeller. There are four insertions in the Vps15 propeller (): an extended loop containing a 310
helix between WD repeats 1 and 2 (residues 1110-1121); an extension of strands 3 and 4 of blade 3 (residues 1156-1172); a large loop with 310
helical twists within the sixth WD repeat (residues 1318-1351), and two additional β-strands within the seventh WD repeat (residues 1385-1426).
Heterotrimeric G protein β subunits form obligate heterodimers with Gγ subunits. The interaction between Gβ and Gγ is largely hydrophobic; the first α-helix of the γ subunit forms a coiled-coil interaction with the Gβ N-terminal α-helix, whereas the second α-helix wraps into a cleft on the bottom surface of the Gβ propeller (36
) (). As noted above, the N-terminus of the Vps15 WD repeat domain is folded against the bottom of the propeller (), and the stretch of N-terminal residues immediately before the propeller partially occlude a potential Gγ binding surface. However, inspection of the hydrophobic regions on the bottom surface of the propeller (upon removal of the N-terminus) of Vps15 indicates there is no equivalent binding cleft for interaction with a Gγ subunit. This finding provides an explanation for our ability to express, purify and crystallize the Vps15 WD domain in the absence of an associated Gγ.
The G protein α subunit typically interacts with Gβγ through two interfaces, termed the “switch interface” and the “N-terminal interface” (39
). The interaction at the switch interface involves residues in and around switch I and switch II of Gα as well as conserved hydrophobic residues on the top surface of the Gβ propeller (Tyr 59, Trp 99, Met 101 and Leu 117 in Gβ1
). The N-terminal interface is formed by interaction of the Gα N-terminal α-helix with four conserved residues in blade 1 of the Gβ propeller (Leu 55, Lys78, Ile 80 and Lys 89 in Gβ1
). The Vps15 propeller provides neither of these binding surfaces for interaction with a Gα subunit. The region that corresponds to the switch interface is comprised of polar amino acids (Thr 67, Thr 113, Thr 116 and Lys 131). Further, superposition of the Vps15 structure on the Gαβγ heterotrimer suggests the Vps15 residues corresponding to the N-terminal interface (Glu 62, Val 87, Lys 89 and Ser 104) would not form a tight interaction with the Gα N-terminal α-helix (not shown). The inability of the Vps15 WD domain to form these interactions, or the absence of a corresponding Gγ subunit, may explain our failure to isolate a stable heterodimeric complex of Gpa1 with the WD domain-containing fragment of Vps15 (by size exclusion chromatography, not shown).
The data presented above reveal the absence of a canonical Gα-binding interface within the Vps15 WD domain. Thus we considered whether other domains of Vps15 might contribute to Gpa1 interactions. In order to determine the possible binding role for these other domains, we co-expressed Gpa1 with several truncations of Vps15 including (a) the kinase domain alone, (b) the kinase domain with the intermediate domain, (c) full length Vps15, (d, e) the intermediate domain with the WD domain, and (f, g) the WD domain alone (). Whereas Gpa1 was tagged with the myc epitope (located internally to preserve function), variants of Vps15 were tagged at the N- and C-terminus with the Flag epitope. We eliminated from consideration any Flag-tag fusions that did not express. We then immunoprecipitated Vps15 and monitored the presence of co-precipitating Gpa1 by immunoblotting. Differences in the apparent abundance of Vps15 truncations could reflect differences in actual abundance or more likely differences in the position of the epitope tag (compare lanes f and g, containing Vps15WD-Flag and Flag-Vps15WD respectively) as well as the size of the tagged protein (which could affect the protein transfer and immunoblot detection). As shown in , the WD domain of Vps15 is sufficient to bind Gpa1, while the kinase domain alone failed to interact. However the kinase domain with the intermediate domain was capable of binding to Gpa1. These data indicate that the intermediate domain, in addition to the Gβ-like WD domain, of Vps15 contributes to the stable interaction with Gpa1.
The WD domain of Vps15 is sufficient but not necessary to bind Gpa1
Vps15 is known to exist in a stable multi-protein complex that includes Atg14 or Vps38 (UVRAG in animals), as well as Vps34 and Vps30 (Beclin-1 in animals) (40
). Atg14 has sequence similarity to canonical Gγ proteins (5
). Thus, we investigated if the Vps15 Gβ-like domain binds to the Gγ-like protein Atg14. To this end we immunoprecipitated each of the Vps15 truncation mutants and monitored copurification of Atg14 by immunoblotting, as described above for Gpa1. As shown in , the WD domain of Vps15 is indeed sufficient to bind to Atg14. The kinase domain alone was also capable of binding to Atg14 (). Typical G protein α subunits bind to the Gγ subunit indirectly, through the common binding partner Gβ. Likewise, Gpa1 bound to the Vps15 WD domain even in the absence of Atg14 expression (). Taken together these data indicate that the Vps15 Gβ-like domain exists in a multi-subunit complex with the Gα and Gγ-like proteins.
The WD domain of Vps15 is sufficient but not necessary to bind Atg14
Atg14 is not necessary to mediate the interaction of Vps15 and Gpa1
Although the crystal structure of the WD-like domain of Vps15 shows that it is comprised of a seven-bladed propeller structure, similar to that of canonical Gβ proteins, this analysis also revealed a lack of conservation in the predicted Gα-binding interface. The lack of a canonical Gα-binding site within Vps15 indicates that interaction must occur in a distinct manner. Accordingly we undertook a molecular evolution analysis to identify potential binding residues within Vps15. We first generated a multiple-sequence alignment of Vps15 orthologs from several fungi, arthropods, mammals and amphibians (Supplemental Data). Invariant and highly conserved residues within this alignment were placed in a spatial context by mapping them onto the crystal structure of Vps15 (far right panel of ). This analysis revealed two residues, Arg-1261 and Phe-1262, that are invariant across all orthologs, implying a strong evolutionary pressure to preserve function. In addition, the side chains protrude into the solvent, making them available for protein-protein interactions. Two additional invariant residues were found on the opposite side of the Vps15 propeller, Glu-60 and Ser-104. However these two residues form multiple hydrogen bonds to each other and may therefore be important for structural constraints. We observed no other invariant residues on the surface of Vps15.
To determine the importance of the conserved Arg-1261, we examined the functional consequences of replacing this residue within Vps15. For these experiments we purified Gpa1 fused to glutathione S-transferase (GST) and monitored co-purification of Vps15 or various Vps15 truncations. Gpa1-GST was used in this case to corroborate the results of the Vps15-immunopreciptation experiments shown above. Additionally, the GST purification method is in our experience better suited for analysis of nucleotide-dependent interactions of G protein α and βγ subunits. Accordingly, the WD domain of Vps15 was co-expressed with Gpa1-GST or with a second G protein α subunit Gpa2-GST, used here as a negative control (). Cell lysates were mixed with glutathione-Sepharose resin, washed extensively, eluted with reduced glutathione, and analyzed by immunoblotting using antibodies against GST (to detect purified Gpa1 or Gpa2) and Myc (to detect any co-purifying Vps15). Although Ste4 was tagged with Myc, it did not bind well with an anti-Myc antibody, and was instead detected using an anti-Ste4 antibody (data not shown, see ). As shown in , the Vps15 WD domain copurified with Gpa1 but did not copurify with Gpa2.
Arg-1261 is necessary for the WD domain of Vps15 to bind efficiently to Gpa1
Arg-1261 is not necessary for larger truncations of Vps15 to bind to Gpa1
We then investigated the functional role of the conserved Arg (). This residue was of particular interest because of a similarly-conserved arginine “finger” present in most G proteins (46
). In G protein α subunits the arginine finger acts in cis
to stabilize the transition state, promote GTP hydrolysis, and turn off the transmitted signal (46
). Thus we substituted Arg-1261 in both a conservative (to Lys) and non-conservative (to Ala) manner, and determined the ability of each mutant to bind to Gpa1 as described above. As shown in , substitution of Arg-1261 to either Lys or Ala resulted in diminished co-purification with Gpa1-GST. This result indicates that Arg-1261 is necessary for Vps15 to bind efficiently to Gpa1. These mutants are likely to be properly folded given that other functions are preserved (see below).
We have shown previously that Vps15 is needed for proper pheromone responses, including pheromone-dependent MAP kinase activation, gene transcription, and cell-division arrest. Thus we investigated the role of Arg-1261 in pheromone signaling. To monitor MAP kinase activity, cell extracts were analyzed by immunoblotting using antibodies against the dually phosphorylated, fully active forms of Fus3 and Kss1 (54
). As shown previously, cells lacking VPS15
exhibit diminished Fus3 phosphorylation after pheromone stimulation; in contrast, cells expressing Vps15R1261A
exhibit wildtype levels of MAP kinase phosphorylation (). To monitor transcriptional activation, we employed a reporter comprised of a pheromone-inducible promoter and β-galactosidase (FUS1-lacZ
). As shown previously, cells lacking VPS15 exhibit dampened induction and a >3-fold rightward shift in the EC50
for pheromone stimulation, whereas cells expressing Vps15R1261A
exhibited wildtype transcription activity (). Finally using the growth arrest plate assay (halo assay), we monitored the ability of the cells to undergo cell division arrest in response to pheromone. Once again, Vps15R1261A
-expressing cells produced wildtype zones of growth inhibition (). Therefore, while it contributes to Gpa1 binding, Arg-1261 does not contribute to Vps15-mediated signaling.
The conserved Arg-1261 is not necessary for G protein signaling at the endosome
In addition to its role in pheromone signaling, Vps15 has well-established functions in promoting autophagy and at least two types of protein trafficking (6
). First, Vps15 is needed for newly synthesized vacuolar proteins, like carboxypeptidase Y (CPY), to move from the endoplasmic reticulum, through the Golgi, to the endosome, and finally to the vacuole compartment. During transport the newly-synthesized CPY becomes modified in the ER to the pro-CPY1 form (67 kDa), then processed in the Golgi to the pro-CPY2 form (69 kDa), and finally it is modified in the vacuole to yield the mature protein product (61 kDa) (10
). To determine if Arg-1261 has any role in CPY sorting, we prepared extracts from vps15R1261A
cells, treated with or without pheromone, and monitored the maturation of CPY by immunoblotting. As shown in , vacuolar sorting of carboxypeptidase Y is unaffected by substitution of Arg-1261 in Vps15.
We have shown above that the WD domain of Vps15 is sufficient to bind Gpa1 in vitro, and does so in a manner that depends on Arg-1261. Nevertheless, Vps15 is capable of promoting the G protein signal in vivo even when Arg-1261 is mutated. Based on these findings we postulated that the kinase and/or intermediate domains of Vps15 might contribute more substantially to the signaling or trafficking functions of the protein. For instance we showed above that the Vps15 intermediate domain as well as the WD domain contributes to Gpa1 binding. Thus we investigated the functionality of Arg-mutant forms of Vps15 containing the intermediate domain as well as the WD domain. The wildtype and arginine-substituted forms of Vps15 were co-expressed with either Gpa1-GST or Gpa2-GST, purified, and analyzed by immunoblotting using antibodies against GST and Myc (to detect any co-purifying Vps15). In this case, Gpa1 bound equally well to wildtype and mutant forms of Vps15 (). These findings indicate that the intermediate domain can compensate for the absence of the conserved arginine present in the WD domain, at least with respect to Gpa1 binding. Our hypothesis was that Arg-1261 functions in the manner of an arginine finger, to promote G protein binding and GTP hydrolysis. However we have shown that this residue is a relatively minor contributor to binding. It remains to be determined if the adjacent conserved Phe-1262 likewise contributes to binding.
Previously, we showed that Vps15 binds preferentially to the unactivated, GDP-bound, form of Gpa1 (5
). Thus we sought to determine if Arg-1261 contributes to the nucleotide-dependent interaction of Vps15 with Gpa1. Wildtype and mutant Vps15 were co-expressed with either Gpa1-GST or Gpa2-GST, then lysed in the presence of either GDP or GDP plus AlF4-
, a transition state mimic that induces the activated conformation of Gα. As observed previously for full-length Vps15, the WD domain with the intermediate domain of Vps15 bound preferentially to GDP-bound (unactivated) Gpa1. The arginine mutants likewise bound preferentially to unactivated Gpa1 (). These results indicate that neither the kinase domain nor the conserved Arg-1261 is necessary to maintain the nucleotide-specificity of Vps15 binding to Gpa1.
The data presented above reveal that the intermediate and WD domains of Vps15 both contribute to Gpa1 binding, and are sufficient to confer nucleotide-specificity of interaction. We have shown previously that Vps15 is required for G protein signaling at the endosome (5
). However we have already ruled out a role for the conserved arginine in sustaining the G protein signaling function of Vps15. These findings suggest that domains necessary for binding and for signaling functions may not fully overlap. Thus we investigated which domains of Vps15 contribute to pheromone signaling, using the same signaling assays described above in . First we determined which domains of Vps15 are necessary to confer MAP kinase activation. As shown in , the kinase domain with the intermediate domain is sufficient to sustain MAP kinase activation in response to pheromone. Likewise, by the transcription-reporter assay the kinase domain with the intermediate domain is sufficient to confer wildtype transcriptional activation () and growth-inhibition responses (). Finally, the kinase domain with the intermediate domain of Vps15 is sufficient to sustain at least some degree of carboxypeptidase Y maturation (). Neither the WD domain alone or the WD domain with the intermediate domain is sufficient to sustain the signaling functions of Vps15 ().
The kinase domain with the intermediate domain of Vps15 is necessary to promote G protein signaling at the endosome
Taken together the findings presented above indicate that the WD domain of Vps15 has a Gβ-like structure and is sufficient to bind Gpa1, but cannot by itself sustain signaling. Whereas the conserved arginine contributes to Gpa1 binding, it is not required for Gpa1 signaling. By comparison, the intermediate domain also contributes to Gpa1 binding, and together with the kinase domain is both necessary and sufficient for pheromone signaling and vacuolar sorting activities.