Vps30p/Apg6p is required for both autophagy and CPY sorting (Kametaka et al. 1998
). However, it was not yet known why and how Vps30p participates in these different membrane trafficking pathways. Recently, it was revealed that PtdIns 3–kinases also function both in autophagy (Kiel et al. 1999
; Petiot et al. 2000
) and protein transport to the vacuole/lysosome (Robinson et al. 1988
; Herman and Emr 1990
; Brown et al. 1995
; Davidson 1995
). Here, we provide evidence that Vps30p functions as a subunit of two distinct large PtdIns 3–kinase complexes: complexes I and II. Each complex contains a specific component, Apg14p (complex I) or Vps38p (complex II) together with three common proteins—Vps34p, Vps15p, and Vps30p. Gene disruption of one of the complex I components resulted in defects in autophagy, whereas gene disruption of one of the complex II components resulted in missorting of CPY. These results indicate that complexes I and II function in autophagy and CPY targeting, respectively. Vps10p is a late-Golgi transmembrane protein that acts as the sorting receptor for CPY (Marcusson et al. 1994
). Mutation in VPS30
changes the subcellular distribution of Vps10p, resulting in a shift of Vps10p from the Golgi to the vacuolar membrane (Seaman et al. 1997
). From these results, Seaman et al. 1997
proposed that Vps30p functions at the step essential for recycling of the Vps10p receptor from the endosome/PVC to the late-Golgi. However, it is still possible that Vps30p (complex II) functions in the anterograde transport of Vps10p–CPY-containing vesicle.
Vps38p could be coimmunoprecipitated with Vps30p in the absence of other factors ( E). Only Vps30p is required for stabilization of Vps38p ( E). These results suggest that Vps38p binds directly to Vps30p (). In contrast, although Vps15p and Vps34p were present in the Δvps38
mutant ( and ), they could not be coprecipitated with Vps30p ( and ). Therefore, it seems that the interaction between Vps30p and the Vps34p–Vps15p core is not direct but mediated by Vps38p in complex II (). Apg14p is unstable in Δvps30
, and Δvps34
cells. These results suggest that both Vps30p and Vps15p–Vps34p may directly bind to Apg14p and conceal recognition sites for proteases (proteolytic systems) in Apg14p or induce Apg14p to adopt a protease-resistant conformation. Thus, Apg14p and Vps38p may act as connectors between Vps30p and Vps15p–Vps34p in complexes I and II, respectively (). However, deletion of APG14
appeared to have no effects on the interaction between Vps30p and Vps15p–Vps34p ( and ) and on the subcellular distribution of Vps30p ( A) and Vps34p ( C), whereas deletion of VPS38
had dramatic effects on them ( and ; and ). These results suggest that complex I may represent only a minor population of PtdIns 3–kinase. In fact, the overall cellular amount of Apg14p is very low; we estimated that wild-type yeast cells contain ~15-fold less Apg14p than Vps30p (data not shown). Moreover, the PtdIns 3–kinase activity of complex I was lower by ~10-fold than that of complex II ( B). Therefore, the effects of the absence of Apg14p might be hidden by the abundant complex II. Although Apg14p and Vps38p have no significant sequence similarities, PairCoil (Berger et al. 1995
) predicted that both proteins have potential coiled coil structures, which often mediate protein–protein interactions.
Figure 9 Model for two distinct PtdIns 3–kinase complexes. Vps15p is anchored to membrane by myristic acid attached to the NH2 terminus of Vps15p (Herman et al. 1991b). Apg14p and Vps38p act as connectors between Vps30p and Vps34p. Phosphorylation of Vps34p (more ...)
To obtain information about the molecular size of the Vps34 PtdIns 3–kinase complexes, gel filtration experiments were performed. When lysates were applied to a Superose 6 column, Vps30p, Apg14p, Vps34p, and Vps38p coeluted in a peak corresponding to ~550 kD (data not shown). However, we could not detect Vps15p in any fractions because Vps15p was somewhat unstable in cell lysates and was gradually degraded by unknown proteases (data not shown). The 550-kD peak might be composed of a mixture of complexes I and II, both lacking Vps15p, that is, Vps30p–Apg14p–Vps34p and Vps30p–Vps38p–Vps34p. Although the artificial instability of Vps15p in vitro made it impossible to estimate the precise molecular weight of complex I and II, it provided two valuable insights into the complex formation. First, Apg14p and Vps38p connector molecules may directly bind to Vps34p. In the vps15 kinase-negative mutant, Vps34p was not coimmunoprecipitated with Vps30p (); that is, Vps34p could not bind to Vps38p, indicating that phosphorylation is required for the binding. From these results, together with the results of gel filtration, we derived the second conclusion: phosphorylation of Vps34p, but not the presence of Vps15p, may be required for the Apg14p–Vps34p and Vps38p–Vps34p interactions (). One attractive hypothesis is that Vps15p-mediated phosphorylation controls binding of Vps34p to Vps38p and Apg14p.
What is the function of PtdIns(3)P? One possibility is that PtdIns(3)P designates the vesicles that are not to be sorted away to the exocytic default pathway. PtdIns(3)P binding proteins may have important roles in presenting PtdIns(3)P as a marker molecule. The FYVE domain, a subfamily of the cysteine-rich RING motif, has been shown to bind directly to PtdIns(3)P (Burd and Emr 1998
). In the yeast S. cerevisiae
, five proteins are known to possess the FYVE domain. One of them, Vac1p, is involved in the fusion between the vesicle derived from the late-Golgi with the endosome/PVC (Peterson et al. 1999
; Tall et al. 1999
). Another FYVE domain–containing protein, Vps27p, is classified as a class E protein (Raymond et al. 1992
). Mutations in class E VPS
genes lead to an accumulation of vacuolar, endocytic, and late-Golgi markers in an exaggerated endosome/PVC, the class E compartment (Raymond et al. 1992
; Piper et al. 1995
). Vps27p may be required for delivery of proteins from the endosome/PVC: multivesicular body formation to the vacuole and for endosome/PVC-to-Golgi retrograde transport.
Alternatively, it is possible that PtdIns(3)P has a role in cargo selection at the vesicle budding step. In this model, binding of a cargo protein to the lumenal domain of the receptor transduces a signal through a conformational change that promotes receptor association with and/or activation of the Vps15p protein kinase. Activation of Vps15p leads to activation of the Vps34 PtdIns 3–kinase. Vps34p-mediated PtdIns(3)P production may recruit effector proteins that function in budding. In mammalian cells, PtdIns(3)P has been shown to play a role in adaptor (AP-2 and arrestin) incorporation into plasma membrane clathrin–coated pits (Gaidarov and Keen 1999
; Gaidarov et al. 1999
). Thus, only lipids surrounding the cargo receptor complex can bud, producing cargo-concentrated vesicles. It is also possible that PtdIns(3)P is required for vesicle formation by generating a driving force to curve the membrane into a bud by repulsive forces between the highly negative polar heads of PtdIns(3)P. However, wortmannin, a phosphoinositide 3–kinase inhibitor, did not inhibit the formation of TGN-derived vesicles but reduced the amount of receptor recruitment into those vesicles in mammalian cells (Gaffet et al. 1997
). This observation is consistent with the idea that PtdIns(3)P is required for cargo selection but not for vesicle formation. In the case of autophagy and the Cvt pathways, complex I might act to load proteins essential for autophagosome/Cvt vesicle formation into vesicles, although it is not yet known if the constituent membranes and proteins of autophagosome/Cvt vesicles are supplied by vesicles.
Interestingly, the Δvps15
mutants have additional phenotypes beyond that of the Δvps30
mutant—impairment of PrA and PrB targeting and growth defects at 37°C. These results indicate that a fraction of the Vps34p–Vps15p complexes function independent of complexes I and II. Growth defects at high temperatures may be caused by defects in endocytosis because most of endocytosis mutations (end
) confer a temperature-sensitive growth defect and VPS34
is allelic to END12
(Munn and Riezman 1994
). It is attractive to speculate that Vps34p–Vps15p forms additional complexes with unknown factors to function in anterograde transport from the late-Golgi to the PVC/endosome for sorting of PrA and PrB and in endocytosis. PtdIns 3–kinase assays using cell lysates showed that deletion of either VPS30
caused only an ~20% reduction of PtdIns 3–kinase activity and the APG14
disruptant showed equivalent PtdIns 3–kinase activity to wild-type cells (). Although it is possible that the postulated additional Vps34p–Vps15p complexes possess significant PtdIns 3–kinase activity, we propose instead that the roles of Vps30p, Apg14p, and Vps38p are not to activate Vps34p but to confer specificities on Vps34p for functions and/or locations where PtdIns(3)P should be produced. Consistent with this proposal, the absence of Vps30p or Vps38p caused some Vps34p to shift from the LSP membrane to the HSP membrane ( C). Further work is needed to determine the precise compartments where complexes I and II act.