Nvj1p is an integral membrane protein of the outer nuclear membrane that creates a platform for the assembly of a unique subdomain of the perinuclear ER membrane. We previously reported that Nvj1p binds and recruits the vacuolar membrane-associated protein Vac8p, which gives rise to Velcro-like interorganelle junctions between the nucleus and vacuole (Pan et al., 2000
). Nvj1p also binds and sequesters the oxysterol-binding protein homolog Osh1p, which is targeted to NV junctions from cytosolic and Golgi pools (Levine and Munro, 2001
; Kvam and Goldfarb, 2004
). In this study, we show that the accumulation of Tsc13p at NV junctions, first reported by Kohlwein et al
), is mediated by a physical interaction with Nvj1p. As in the cases of Osh1p and Vac8p, the localization of Tsc13p to NV junctions is dependent on Nvj1p, and its sequestration into NV junctions is directly proportional to NVJ1
expression levels. Moreover, we demonstrate that Nvj1p binds and recruits Tsc13p to the perinuclear ER from surrounding ER compartments even in cells lacking Vac8p and NV junctions. Therefore, the vacuole membrane is not required for the sequestration of Tsc13p into Nvj1p-enriched subdomains of the perinuclear ER.
The enrichment of both Tsc13p and Osh1p at NV junctions is reminiscent of the accumulation of factors involved in lipid biochemistry at other ER contact sites (Stone and Vance, 2000
; Pichler et al., 2001
). Studies of these contact sites suggest that interorganelle molecular junctions might be a general property of most ER contact sites (reviewed in Levine, 2004
). However, the Velcro-like Vac8p-Nvj1p interaction is the only known interorganelle junction-forming apparatus. Uniquely, NV junctions are also platforms for the PMN, during which portions of the nucleus are pinched off into the vacuole lumen and degraded. Because NV junctions strictly define the portion of the nuclear envelope engulfed by the invaginating vacuole membrane, Nvj1p and all three of its known binding partners (Vac8p, Tsc13p, and Osh1p)— and other associated proteins or lipids—must be delivered by PMN into the hydrolytic vacuole lumen.
Because Nvj1p expression levels are tightly coupled to the formation and proliferation of NV junctions and PMN, the Nvj1p-dependent targeting of Tsc13p to NV junctions is spatially and temporally correlated with PMN biogenesis. In addition to demonstrating a physical interaction between Tsc13p and Nvj1p, we present three lines of evidence in support a functional relationship between the targeting of Tsc13p to NV junctions and the biogenesis of PMN vesicles. First, morphometric analyses demonstrate that the lumenal diameters of PMN structures are significantly smaller in tsc13-1 and elo3-Δ tsc13-1 fatty acid elongation mutants compared to wt cells. Second, PMN blebs and vesicles are strikingly smaller in starved cells after treatment with cerulenin, an inhibitor of de novo fatty acid synthesis and elongation. Last, cerulenin promotes the marked delocalization of Tsc13p-EGFP from NV junctions in starved cells, which suggests that a continuous supply of fatty acid substrates may be required for the proper targeting of Tsc13p to NV junctions. Both consequences of cerulenin treatment (i.e., the stunting of PMN structures and the mistargeting of Tsc13p-EYFP from NV junctions) are suppressed by feeding the cells with myristic acid, which bypasses the cerulenin-induced block in de novo fatty acid synthesis. These observations suggest that the enzymatic activity of Tsc13p during VLCFA biosynthesis contributes to the biogenesis of PMN blebs and vesicles at NV junctions.
The delocalization of Tsc13p-EGFP from NV junctions in the presence of cerulenin is interesting because it links the enzyme's localization to the supply of its fatty acid substrates. By analogy, the localization of yeast acyl chain desaturase, Ole1p, was reported to shift from a uniform ER distribution to punctate domains along the cell periphery in a substrate-dependent manner (Tatzer et al., 2002
). Because Tsc13p physically associates with Elo2p and Elo3p fatty acid elongases (Kohlwein et al., 2001
), the substrate-dependent localization of Tsc13p to NV junctions implies that active elongation complexes may transiently assemble at NV junctions. Thus, distinct elongation complexes containing Tsc13p are likely required for the normal biogenesis of PMN blebs and vesicles. In support of this, we determined that additional enzymes of the microsomal VLCFA elongation system (Elo2p, Elo3p, and YBR159p) are present, but not concentrated, within NV junctions. Moreover, the lumenal diameters of PMN structures are significantly smaller in elo3-
cells than tsc13-1
Despite the dramatic effects of cerulenin on the size of PMN blebs and vesicles, cerulenin showed no significant effects on PMN-mediated protein turnover during starvation. This observation demonstrates that the rate of PMN throughput is not necessarily proportional to the size of PMN intermediates. Wild-type rates of PMN are probably maintained in the presence of cerulenin through the formation of many small and/or unstable PMN blebs and vesicles. Under these conditions, the scission of PMN blebs into the vacuole lumen may occur faster than normal due to structural instabilities in the membrane architecture of PMN blebs. Alternatively, we cannot rule out the possibility, however unlikely, that components of NV junctions may be degraded by other proteolytic systems under these conditions. This issue deserves more study because the packaging of some nuclear constituents into PMN vesicles, such as the granular nucleolus, might be affected by altering the size of PMN blebs.
Previously, we reported that cells depleted of the entire oxysterol-binding protein family (Osh1p-Osh7p) accumulate PMN intermediates, consistent with the idea that sterol lipid homeostasis is required for the proper formation of mature blebs and vesicles (Kvam and Goldfarb, 2004
). In addition to sterol lipids, our present study suggests a role for VLCFA-containing lipids at NV junctions. Several studies in yeast have highlighted an importance for VLCFA synthesis in membrane structure and vesicular trafficking. For example, VLCFA synthesis is integral to the structure of the yeast nuclear envelope (Schneiter et al., 1996
) as well as for vacuole assembly and fusion (Faergeman et al., 2004
). Spontaneous mutations in either of the yeast VLCFA elongases ELO2
bypass the requirement for vesicular soluble N
-ethylmaleimide sensitive factor attachment protein receptors during secretion (David et al., 1998
). VLCFAs are proposed to facilitate these roles by promoting the formation of highly curved membrane structures (reviewed in Schneiter and Kohlwein, 1997
). In model membranes, VLCFA-substituted lipids have been shown to perturb bilayer structure and facilitate membrane curvature due to their extended hydrophobic tails (Schneiter and Kohlwein, 1997
). Thus, deficiencies in VLCFA-substituted lipids are associated with circumstances of membrane hyper-rigidity, such as restrictive dermopathy in mammals (Herrmann et al., 2003
). Based on these observations, we propose that VLCFAs may be required for the efficient biogenesis of highly curved blebs and vesicles during the process of PMN. It is important to note that PMN blebs and vesicles are comprised of three concentric membranes, namely, the vacuole membrane and the double-membrane nuclear envelope, which pose unusual constraints on their biogenesis.
Interestingly, myriocin, which inhibits the biosynthesis of ceramides and sphingolipids, failed to alter PMN morphology in starving cells. These data imply that VLCFAs alone, or VLCFA-containing lipids other than ceramides and sphingolipids, play a specific role during PMN. By analogy to the role of sphingolipids in the partitioning of membrane proteins into lipid rafts (reviewed in Salaun et al., 2004
), the accumulation of VLCFAs and/or VLCFA-containing lipids in NV junctions could serve to facilitate the partitioning of Nvj1p and associated binding partners (including Tsc13p itself) into NV junctions. However, this model does not exclude a role for ceramides or sphingolipids in the biology of NV junctions, because, in the presence of myriocin, these VLCFA-containing lipids could be recruited into NV junctions from preexisting pools. Rather, our results are consistent with the more general idea that the lipid composition of NV junctions is modified by Tsc13p (and the microsomal VLCFA elongation system) preceding or following the formation of nascent PMN blebs and that this lipid environment contributes to normal PMN biogenesis.
The Nvj1p-mediated targeting of Tsc13p to NV junctions could pose other physiological consequences for the cell. For example, the sequestration of Tsc13p away from peripheral ER pools is likely to reduce the rate of local VLCFA biosynthesis in other subcellular ER compartments, which could have a variety of effects on bulk membrane biology. The degradation of NV junction-associated Tsc13p during PMN may also affect the general rate of VLCFA biosynthesis in the cell during starvation. Because VLCFA-containing lipids are required for vacuole biogenesis and fusion (Faergeman et al., 2004
), their production in the perinuclear ER at NV junctions could provide a ready source of lipids for vacuole compartments.
In conclusion, this study demonstrates that Nvj1p binds and sequesters Tsc13p, an essential enoyl-CoA reductase in the microsomal fatty acid elongation system, into NV junctions through a mechanism directly analogous to the Nvj1p-mediated targeting of Vac8p and Osh1p (Kvam and Goldfarb, 2004
; Pan et al., 2000
). By analogy to other ER membrane contact sites, these findings support a role for Nvj1p in the organization of a specialized domain of the nuclear ER that mediates PMN. Our results argue most strongly that Tsc13p plays a compartmentalized role in the biogenesis of PMN structures, and that VLCFA-containing lipids contribute to the production of these trilaminar structures. However, by analogy to the utility of ER-mitochondria contact sites in the interorganellar trafficking of phospholipids, it is also possible that the targeting of Tsc13p and Osh1p to NV junctions may function in general lipid metabolism and homeostasis. Further work is needed to distinguish among these interrelated models.