The NV-junction is a zone of contact between vacuoles and the nuclear envelope. It affects membrane organization on the nuclear side because nuclear pore complexes are not found in vicinity of the vacuolar membrane (Severs et al., 1976
; Pan et al., 2000
). Here we show that Vph1p-GFP is excluded from the vacuolar membrane sections that form an NV junction. This barrier is specific for V-ATPase and the exclusion does not affect the VTC complex, which has a cytosolic domain of similar size. It has been speculated that NV junctions might have a special lipid composition (Kvam and Goldfarb, 2006a
). The results we obtained with the mutants in Osh genes and in ceramide metabolism support this notion and suggest a role of lipids in NV junction formation and function. An observation that strengthens this view is that the accumulation of the VTC complex at NV junctions is abolished by inhibition of phosphatidylinositol-3-kinase (C. Dangelmayr, R. Dawaliby et al.
, unpublished results). NV junctions might hence form a specialized lipid zone on the vacuolar membrane that excludes certain vacuolar proteins by providing an unsuitable membrane environment for them. In that sense they resemble lipid rafts, specialized membrane microdomains that have been extensively characterized. Rafts are rich in cholesterol and sphingolipids (Simons and Ehehalt, 2002
; Korade and Kenworthy, 2008
) and serve as organizing centers for the assembly of signaling molecules. They influence membrane fluidity and membrane protein trafficking and regulate neurotransmission and receptor trafficking (Simons and Ehehalt, 2002
; Pichler and Riezman, 2004
). Similarly, NV junctions recruit selected proteins such as Osh1p, Tsc13p, and VTC and exclude others.
V-ATPase exclusion is reverted by concanamycin A. This suggests that NV junctions form a barrier that requires the electrochemical potential of the vacuolar membrane. This potential depends to a large extent on the proton translocation activity of the V-ATPase which acidifies vacuoles (Forgac, 2007
). Numerous studies demonstrated roles of the V-ATPase in membrane homeostasis and vesicular traffic. Only some of these depend on proton pump activity, such as autophagosome-lysosome fusion, passage through endosomes, or the fragmentation of yeast vacuoles (Schmid et al., 1989
; Yamamoto et al., 1998
; Liu et al., 2005
; Baars et al., 2007
; Kawai et al., 2007
). An example for a function independent of proton-translocation is the physical role of the V0
sector during membrane fusion at various membrane trafficking steps (Peters et al., 2001
; Bayer et al., 2003
; Hiesinger et al., 2005
; Liegeois et al., 2006
; Sun-Wada et al., 2006
; Peri and Nusslein-Volhard, 2008
). A purely physical role of the V-ATPase in formation of PMN vesicles is unlikely because the process is sensitive to concanamycin A and because V-ATPase is excluded from PMN structures and NV junctions that give rise to these vesicles. Thus, one should rather consider effects via the membrane potential. Vesicle formation and fission can depend on lateral inhomogeneities within membranes, induced either by spontaneous phase separations of lipids (Julicher and Lipowsky, 1993
; Sackmann and Feder, 1995
; Julicher and Lipowsky, 1996
; Munn et al., 1999
; Proszynski et al., 2005
; Falguieres et al., 2009
), or by the concentration of membrane-apposed or membrane-integral proteins (Wenk and De Camilli, 2004
; Lee et al., 2005
; Ramos et al., 2006
). Such lateral phase separations can be strongly influenced by the membrane potential, as exemplified by the massive compartmentation of the yeast plasma membrane into two clearly distinct zones which are occupied either by the plasma membrane ATPase Pma1p or the arginine symporter Can1p (Malinska et al., 2003
). These two zones mix when the membrane is depolarized (Grossmann et al., 2007
). Also, phase separation in synthetic membrane systems can depend on the membrane potential (Herman et al., 2004
; Schaffer and Thiele, 2004
). Thus, our observations and those from previous studies are consistent with the hypothesis that the full differentiation of an NV junction may comprise lipid phase separations depending on the electrochemical potential across the vacuolar membrane.
A comprehensive study systematically tested vacuole- and autophagy-related genes for a role in PMN (Krick et al., 2008
). This confirmed numerous known PMN factors and identified a novel requirement for Atg genes in PMN. Whereas our microscopic observations on atg mutants agree with this study, there is a discrepancy concerning the V-ATPase which Krick et al. (2008
) found not to be required for PMN. This discrepancy could be due to the different assays used. We visualized transfer of the nucleolar protein Nop1p-GFP into vacuoles microscopically. This microscopic assay is independent of the actual degradation of the PMN vesicles or the reporter. In their tests of V-ATPase mutants Krick et al. (2008
) used the proteolytic degradation of a GFP-Osh1p fusion protein as a measure for PMN. The assay is based on the fact that GFP is more protease-resistant than the linker between GFP and Osh1p and hence accumulates in vacuoles. The parameters influencing this assay are complex. On induction of PMN, the GFP-Osh1p fusion is transferred into vacuoles, the PMN vesicle membranes must be degraded, and then the vacuolar proteases cleave GFP-Osh1p and GFP. Although the GFP domain is more protease-resistant than the GFP-Osh1p fusion, it is nevertheless degradable by vacuoles, which can easily be visualized by pulse-chase experiments in which expression of new GFP is prevented by Gal-shut-off experiments (our unpublished results). The relative protease-resistance of GFP leads to its accumulation inside vacuoles to a certain level. This level, however, depends on multiple parameters, such as the protease and lipase activities inside the vacuoles, the time that the fusion protein is exposed to vacuolar hydrolases, the kinetics with which the GFP-Osh1p linker and the released GFP domain are degraded, and the level of PMN activity. Mutations can reduce the activities of vacuolar lipases and proteases to varying degrees (Kane, 2006
; Forgac, 2007
), which can influence the half-lives of GFP-Osh1p and its GFP fragment differently. V-ATPase mutants still show some proteolytic activity in their vacuoles but at much lower levels than wild-type cells. Thus, V-ATPase mutants might still be able to cleave the sensitive GFP-Osh1p linker but degrade the more resistant GFP more slowly than wild types. This can hide a defect in PMN since even if less GFP-Osh1p is transferred into vacuoles delayed degradation of its GFP fragment could lead to its overproportional accumulation. The data of Krick et al.
(C) are consistent with this notion because they show two important differences between V-ATPase mutants and the wild-type: Wild-type cells contain virtually no fragment before induction of PMN. Induction dramatically enhances the transfer of GFP-Osh1p into wild-type cells, producing the fragment. In contrast, the V-ATPase knockout Δvma2 shows high levels of GFP fragment already before induction of PMN and these remain unaltered upon PMN induction. Furthermore, Δvph1 mutants, which retain limited vacuolar acidification due to the presence of the Stv1p-containing V-ATPase complexes (Manolson et al., 1994
), show low levels of GFP fragment before induction. On induction of PMN, GFP fragment accumulates but with a significant delay relative to wild-type cells. The interpretation of the proteolysis-based assay of PMN is hence not straightforward for mutants influencing the hydrolytic capacity of the vacuoles. In these cases the assay must be validated by determining the half-lives of the GFP-fusion and the GFP-fragment in each mutant.
Cells overexpressing N-terminally tagged Nvj1 excluded Vph1p from vacuolar sites that were not adjacent to the nucleus. These random exclusion sites were next to Nvj1p patches but far from the nucleus. This confirms that contact with nuclear material is dispensable for making contacts between vacuoles and the ER or outer nuclear membrane (Roberts et al., 2003
). Furthermore, it shows that differentiation of the contact sites into “NV” junctions (or their ER-equivalents) is independent of the nucleus. That this “differentiation” can be regarded as a separate step in the establishment of NV junctions is supported by the fact that inhibition of V-ATPase pump activity by concanamycin A barely reduces the frequency of nuclear-vacuolar contacts but strongly reduces Vph1p exclusion from these sites ().
Integrating our results with those of earlier studies (Kvam and Goldfarb, 2007
; Krick et al., 2008
), we can hence extend the model of NV junction formation and PMN by a new step. Whereas V-ATPase activity is not required for forming nuclear-vacuolar contact sites, further evolution into NV junctions that generate a diffusion barrier on the vacuolar side does depend on it. This diffusion barrier likely depends on a specific lipidic environment. The deformation of NV junctions into PMN sites and the subsequent formation of vesicles from them requires the electrochemical potential and completion of the process depends on Atg genes.