NV junctions (nucleus–vacuole junctions) in Saccharomyces cerevisiae are MCSs (membrane contact sites) formed through specific interactions between Vac8p on the vacuole membrane and Nvj1p in the outer nuclear membrane, which is continuous with the perinuclear ER (endoplasmic reticulum). NV junctions mediate a unique autophagic process that degrades portions of the yeast nucleus through a process called ‘piecemeal microautophagy of the nucleus’ (PMN). Our studies suggest that the lipid composition of NV junctions plays an important role in the biogenesis of PMN structures. NV junctions represent a unique model system for studying the biology of ER MCSs, as well as the molecular mechanism of selective microautophagy.
The ER (endoplasmic reticulum) is closely associated with a number of functionally distinct organelles, including mitochondria, Golgi, peroxisomes, vacuoles/lysosomes and the plasma membrane (reviewed in [1,2]). Microscopic inspections of these sites show that the ER membrane converges with the membrane of a dissimilar organelle, forming a heterologous MCS (membrane contact site) (reviewed in ). Much of what is known about the function of MCSs has come from studies of MAMs (mitochondria-associated ER membranes) and PAMs (plasma membrane-associated ER membranes), which mediate inter-organelle lipid trafficking (reviewed in ). The fact that MAMs and PAMs can be biochemically purified suggests that junctional apparatus may play important roles in the formation and maintenance of these and other MCSs. Indeed, electron-dense cross-bridges have been observed across the narrow cytoplasmic gaps of ER–peroxisome contact sites , and the formation of MAMs in vitro is inhibited by proteolysis of mitochondrial surface proteins . The NV junction (nucleus–vacuole junction) in yeast is currently the only MCS where the junction apparatus responsible for creating and maintaining a stable heterologous MCS has been identified. Here, we review what is known about the structure and function of NV junctions in Saccharomyces cerevisiae.
NV junctions link two dissimilar organelles
The yeast nucleus, like that of higher eukaryotes, is enclosed by a specialized membrane system called the nuclear envelope, which consists of two concentric bilayers connected at numerous sites by fenestrae called nuclear pores. The ONM (outer nuclear membrane) of the nuclear envelope is continuous with the ER and, as such, is typically studded with ribosomes. Thus the nuclear envelope is considered a functionally distinct subcompartment of the ER (reviewed in ). Thin-section electron microscopy has revealed the existence of extensive contact sites between the nuclear envelope and vacuoles in yeast, which we have named NV junctions [6,7]. Possible analogous associations between lysosomes (the equivalent of vacuoles) and nuclei in higher eukaryotes have been reported .
The yeast vacuole, like the mammalian lysosome, is an acidic organelle that functions in a variety of macromolecule recycling and storage capacities, including autophagy. Autophagy, or ‘self-eating’, is classically viewed as a starvation response, but it is employed in numerous other contexts where bulk degradation of the cytoplasm (including organelles) is required (reviewed in ). In general, cellular autophagy takes place through either macroautophagic or microautophagic routes. During macroautophagy, double-membrane vesicles called autophagosomes enclose and traffic bulk cytoplasm and organelles to the vacuole for degradation . Microautophagy, in contrast, involves the direct sequestration of cargo into an invagination of the vacuole membrane, and therefore lacks vesicular intermediates .
NV junctions expand and proliferate under conditions of nutrient depletion in yeast . We have determined that NV junctions mediate a unique microautophagic process called ‘piecemeal microautophagy of the nucleus’ (PMN) that targets portions of the yeast nucleus for degradation in the vacuole . During PMN, the nuclear envelope and associated nucleoplasmic components, such as nucleolar pre-ribosomes, are sequestered into an invagination of the vacuole membrane, forming a nuclear ‘bleb’ that is pinched-off as a tri-lamellar PMN vesicle into the vacuole lumen and degraded by vacuolar hydrolases (Figure 1) .
Five morphological intermediates of PMN in yeast
NV junctions are stabilized by protein–protein interactions
We unexpectedly discovered the molecular components of yeast NV junctions through studies of the vacuolar membrane protein Vac8p, an Armadillo-repeat protein related to β-catenin and the nuclear transport receptor, importin α . Vac8p is an acylated adapter protein that functions in a variety of vacuole processes, including vacuole inheritance and homotypic fusion [11,12]. Several binding partners of Vac8p were identified by the yeast two-hybrid method, including an uncharacterized protein that was eventually named Nvj1p (NV junction protein) . Biochemical and microscopy studies using GFP (green fluorescent protein)-tagged reporters revealed that Nvj1p is an integral membrane protein of the ONM that interacts through its cytosolic C-terminus with Vac8p [7,13]. Nvj1p–Vac8p complexes form a stable junction between the nucleus and vacuole by clustering into a Velcro-like patch (Figure 1B). Deletion of either NVJ1 or VAC8 precludes the formation of NV junctions . The expansion and proliferation of NV junctions is regulated by the expression of NVJ1, whose promoter contains several stress response elements [10,14]. Overexpression of NVJ1 promotes radical expansion in the surface area of NV junctions and leads to defects in vacuole structure and inheritance, presumably due to the depletion of Vac8p pools . In the absence of Vac8p, Nvj1p spreads over the surface of the ONM but fails to escape into transitional or cortical ER compartments .
NV junctions harbour factors involved in lipid biosynthesis and trafficking
Specific heterologous ER contact sites have been implicated in the synthesis and non-vesicular trafficking of lipids in eukaryotes (reviewed in ). These MCS domains are typically enriched in proteins active in lipid and sterol biosynthesis. Previous studies have demonstrated that NV junctions also accumulate proteins involved in lipid biosynthesis and trafficking [15,16]. One of these proteins, the enoyl-CoA reductase Tsc13p, is an essential polytopic ER membrane protein that functions in the synthesis of VLCFAs (very-long-chain fatty acids) . The other, Osh1p, is related to a large family of putative sterol–lipid transfer proteins bearing similarity to human OSBP (oxysterol-binding protein) . Crystal structures of a close relative to Osh1p (Osh4p/Kes1p) verify that the conserved signature domain of the yeast OSBP family folds into a hydrophobic sterol-binding cavity with a flexible lid that seals sterols away from the aqueous environment . Our studies indicate that Nvj1p is necessary and sufficient to sequester both Osh1p and Tsc13p to the ONM. This sequestration occurs in the absence of Vac8p and without any apparent link between the nucleus and vacuole [13,18].
PMN biogenesis is dependent on activities associated with Tsc13p and Osh1p
It is likely that the lipid composition of NV junctions is modified in order to facilitate the biogenesis of PMN blebs and vesicles. Thus Tsc13p and Osh1p may function from within NV junctions to alter the local lipid composition of the nuclear and vacuole membranes. In support of this hypothesis, cells that are deficient in activities associated with Tsc13p and Osh1p exhibit distinct defects in the biogenesis of PMN vesicles. Specifically, pharmacological inhibition of fatty acid synthesis and elongation, as well as genetic mutations in TSC13, result in dramatic decreases in the size of PMN blebs and vesicles . Moreover, cells that are defective in sterol trafficking as a consequence of deleting the seven-membered yeast Osh family (Osh1p–Osh7p), which show partial functional redundancy , appear to arrest PMN at a late stage . These results indicate that specific lipids associated with Osh1p and Tsc13p probably play important roles in PMN, perhaps by facilitating membrane bending and scission events (Figure 1A). Alternatively, by analogy to MAMs and PAMs, the sequestration of Osh1p and Tsc13p at NV junctions may serve more general roles in cellular lipid homoeostasis. In this case, alterations in PMN biogenesis that are associated with the loss of Osh proteins and Tsc13p activity could be indirect. These two hypotheses are not mutually exclusive.
At present, NV junctions are the only heterologous MCSs with a characterized junctional apparatus. As such, NV junctions have significant potential to serve as a useful paradigm for investigating the structure and function of other MCSs.
Non-Vesicular Intracellular Traffic: Biochemical Society Focused Meeting held at Goodenough College, London, U.K., 15–16 December 2005. Organized and edited by S. Cockcroft (University College London, U.K.) and T. Levine (Institute of Ophthalmology, London, U.K.).