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3622 Research Article

Introduction

The endoplasmic reticulum (ER) is an interconnectedmembrane network composed of structurally distinctcompartments, including the peripheral or cortical ER and theperinuclear ER, which surrounds the nucleus (reviewed inVoeltz et al., 2002). Although these compartments aretopologically continuous and share a common lumen, each hasunique properties and functions (reviewed in Levine andRabouille, 2005; Voeltz et al., 2002). The perinuclear ER,which comprises the nuclear envelope (NE), consists of twoconcentric membrane sheets connected at fenestrae callednuclear pores. The inner nuclear membrane (INM) serves asan attachment sites for chromatin and, in higher eukaryotes, isassociated with the nuclear lamina (reviewed in Hetzer et al.,2005). The outer nuclear membrane (ONM), like the rough ER,is studded with ribosomes but, uniquely, for example, containscytoskeletal attachment sites for nuclear positioning (reviewedin Hetzer et al., 2005). The mechanisms responsible forcreating and maintaining these structurally distinctcompartments are poorly understood.

Accurate protein sorting to the nuclear envelope is criticallyimportant for nuclear function and cell survival. Mutationsaffecting the targeting and function of specific nuclearenvelope proteins have been linked to several human diseasescollectively termed nuclear envelopathies (reviewed in Somechet al., 2005). In general, proteins localize to the nuclearenvelope through associations with resident nuclear proteins

(reviewed in Holmer and Worman, 2001). Sorting to the INM

occurs initially via energy-dependent interactions with thenuclear pore complex (NPC) (Ohba et al., 2004; Beilharz etal., 2003). Nuclear lamins exemplify an important class of peripheral INM proteins that are accompanied across the NPCby soluble receptors called karyopherins (reviewed in Hetzeret al., 2005). Likewise, tail-anchored INM proteins translocatethrough the NPC and insert post-translationally into the INM(Beilharz et al., 2003). However, integral INM proteins that aresynthesized in the extranuclear rough ER are sorted to the INMprobably by diffusion across the continuous membrane of thenuclear pore (Ohba et al., 2004). Saturable interactions withnuclear lamins, chromatin, or other nuclear proteins arethought to retain these proteins within the INM (Ostlund et al.,2006).

Mechanisms governing the sorting of ONM proteins arepoorly characterized, as only a few cases are known. A WPPdomain (tryptophan-proline-proline) at the N-terminus of asubset of plant proteins is necessary and sufficient to target theONM, presumably by interacting with factors on the nuclearsurface (Patel et al., 2004). Additionally, metazoan KASH-domain-containing proteins (Klarsicht, ANC-1, and Synehomology) are retained in the ONM through interactions acrossthe perinuclear lumen with specific INM proteins (reviewed inWorman and Gundersen, 2006; Starr and Fischer, 2005).KASH domains physically bind the luminal SUN domains(Sad1p and UNc-84 homology) of SUN proteins, which are

Nvj1p resides in the outer nuclear membrane (ONM) andbinds the vacuole membrane protein Vac8p to formnucleus-vacuole (NV) junctions in Saccharomyces cerevisiae. The induction of NVJ1 expression duringstarvation results in the sequestration of two additionalbinding partners, Tsc13p and Osh1p. Here, we map thedomains of Nvj1p responsible for ONM targeting andpartner binding. ONM targeting requires both the N-terminal signal anchor-like sequence and the topogenicmembrane-spanning domain of Nvj1p. The N-terminalsignal anchor-like sequence may anchor Nvj1p in the ONMby bridging to the inner nuclear membrane. A regionencompassing the membrane-spanning domain is sufficientto bind Tsc13p. Osh1p and Vac8p bind to distinct regionsin the cytoplasmic tail of Nvj1p. Overexpression of Nvj1p

in trp1 cells causes a growth defect in low tryptophan thatis rescued by additional copies of TAT1 or TAT2 tryptophanpermeases. Conversely, nvj1- trp1 cells grow faster than NVJ1+ trp1 cells in limiting tryptophan. Importantly,deleting the Osh1p-binding domain of Nvj1p abrogates thetryptophan transport-related growth defect of Nvj1p-overexpressing cells. Therefore, the Nvj1p-dependentsequestration of Osh1p negatively regulates tryptophanuptake from the medium, possible by affecting thetrafficking of tryptophan permeases to the plasmamembrane.

Key words: Nuclear envelope, Endoplasmic reticulum, Nucleus-

vacuole junctions, Tryptophan permease, Osh1p, Tsc13p, Nvj1p

Summary

Structure and function of nucleus-vacuole junctions:

outer-nuclear-membrane targeting of Nvj1p and a role

in tryptophan uptake

Erik Kvam and David S. Goldfarb*

Department of Biology, University of Rochester, Rochester, NY 14627, USA*Author for correspondence (e-mail: [email protected])

Accepted 9 June 2006 Journal of Cell Science 119, 3622-3633 Published by The Company of Biologists 2006 doi:10.1242/jcs.03093

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3623Functional domains of Nvj1p

anchored to the INM by nuclear lamins (Crisp et al., 2006).Together, KASH and SUN proteins mediate the assemblyof large nuclear envelope-associated complexes (LINCcomplexes) that physically link the cytoskeleton tonucleoplasmic components.

By analogy to KASH proteins, Nvj1p is a yeast membraneprotein found exclusively in the ONM where it interacts with

Vac8p on the vacuole membrane to form nucleus-vacuole (NV) junctions (Pan et al., 2000). In the absence of Vac8p, Nvj1plocalizes evenly over the surface of the nuclear envelope butfails to escape into the cortical ER, even when overexpressed(Pan et al., 2000). Thus, an undetermined mechanism mediatesthe strict localization of Nvj1p to the ONM. Aside from Vac8p,Nvj1p also sequesters two proteins with roles in lipidhomeostasis, Osh1p and Tsc13p, to the nuclear surface (Kvamet al., 2005; Kvam and Goldfarb, 2004). The fraction of Osh1por Tsc13p present at NV junctions depends on the expressionlevel of Nvj1p, which is upregulated in response to nutrientdepletion. Tsc13p is an essential ER membrane proteininvolved in the synthesis of very-long-chain fatty acids(VLCFAs), which are important constituents of ceramides,

sphingolipids, GPI anchors and unusual inositolphospholipidsand, as such, play important roles in membrane fluidity, lipidraft biogenesis, and cell signaling (Eisenkolb et al., 2002;Kohlwein et al., 2001; Dickson, 1998). Osh1p, on the otherhand, is a member of a large family of eukaryotic oxysterol-binding protein-related proteins (ORPs) that share a conservedoxysterol-binding domain, which forms a hydrophobic sterol-sensing cavity (Im et al., 2005). Yeast lacking the overlappingOsh protein family (Osh1p-Osh7p) show significant defects insterol-dependent processes including endocytosis, vesicletransport, and homotypic vacuole fusion (Beh and Rine, 2004).Consistent with a role in sterol regulation, osh1- cells exhibita cold-sensitive tryptophan transport defect akin to erg6 mutants that are defective in ergosterol synthesis (Levine andMunro, 2001; Jiang et al., 1994). Ergosterol is required fortrafficking of the high-affinity tryptophan permease, Tat2p,to the plasma membrane via detergent-resistant membranedomains (Umebayashi and Nakano, 2003). The localization of Osh1p is regulated by several targeting determinants, includinga PH domain specific for Golgi membranes (Levine andMunro, 2001), a FFAT motif (FF in an acidic tract) that targetsthe ER through associations with Scs2p (Loewen et al., 2003),and an N-terminal ankyrin repeat domain that mediateslocalization to NV junctions (Levine and Munro, 2001).

NV junctions are sites of a starvation-induced autophagicprocess called piecemeal microautophagy of the nucleus(PMN) that degrades portions of the yeast nucleus in thehydrolytic vacuole lumen (Roberts et al., 2003). During PMN,the vacuole membrane envelopes a section of the nuclearenvelope through invagination, forming a nuclear ‘bleb’.Ultimately, this tri-laminar PMN bleb, consisting of the inner-and outer-nuclear membranes and the vacuole membrane, ispinched-off as a vesicle and degraded in the vacuole lumen(Roberts et al., 2003). PMN appears to respond to nutrientstarvation by scavenging and recycling nonessential nuclearcomponents, including portions of the nuclear envelope and,often, underlying nucleolar pre-ribosomes (Roberts et al.,2003). Activities associated with Tsc13p and the Osh-proteinfamily are required for efficient PMN biogenesis (Kvam et al.,2005; Kvam and Goldfarb, 2004).

In this study, we map the sequences of Nvj1p that mediatesorting to the ONM and associations with Vac8p, Tsc13p andOsh1p. The N-terminal signal anchor-like sequence of Nvj1p isnot required for either targeting to the ER or correct orientationin the membrane, but is required for ONM anchoring, possiblyby bridging to the INM. The membrane-spanning domain of Nvj1p mediates membrane insertion and also interacts with

Tsc13p. Osh1p and Vac8p bind to non-overlapping regions inthe cytoplasmic C-terminus of Nvj1p. Based on growthphenotypes associated with the Osh1p-Nvj1p interaction, wepresent evidence that Nvj1p functions as a negative regulator of tryptophan transport during nutrient depletion.

ResultsSaccharomyces orthologs of Nvj1p share fourconserved domainsNvj1p is an integral ER membrane protein that localizesexclusively to the ONM and sequesters proteins from diversecellular pools to the nuclear surface. Since Nvj1p lacks knownsequence motifs, we identified functionally constrained regionsin Nvj1p by comparing orthologous protein sequences from six

Saccharomyces species, including four ‘stricto senso’ speciessimilar to S. cerevisiae (S. paradoxus, S. mikatae, S. bayanusand S. kudriavzevii) and two more divergent species (S.castellii and S. kluyveri) (Cliften et al., 2001). Thesealignments revealed four conserved regions in Nvj1p (Fig.1A,B). Region I at the N-terminus is characterized by ahydrophobic signal anchor-like sequence (Fig. 1B,C). RegionII overlaps with the predicted membrane-spanning domain of Nvj1p (Fig. 1B,C). Region III is adjacent to the membrane-spanning domain, and region IV maps to the C-terminal end of Nvj1p (Fig. 1B). These conserved regions helped guide ourefforts in mapping the putative functional domains of Nvj1p.

Region IV is the Vac8p-binding domain of Nvj1pProtein-protein interactions between Nvj1p and Vac8p arerequired for the formation of NV junctions. These interactionsare remarkable because they represent the only currentlyknown inter-organelle junction apparatus in eukaryotes(Levine, 2004). Previously, we showed that Vac8p co-purifieswith Nvj1p and genetically interacts with the C-terminal tail of Nvj1p (aa 261-321) by yeast two-hybrid analysis (Pan et al.,2000). These results position the Vac8p-binding domain of Nvj1p within the vicinity of region IV (Fig. 1A,B). Todetermine whether region IV is necessary for an associationwith Vac8p, we deleted a cluster of conserved residues at theC-terminal end of Nvj1p (aa 292-321; Fig. 1A,B) and analyzedthe localization of the truncated reporter (Nvj1p(293-321)-EGFP) in the presence and absence of Vac8p. Normally inVAC8+ cells, full-length Nvj1p-EGFP localizes to patches onthe nuclear surface that co-localize with FM4-64-stainedvacuole membranes, producing a yellow fluorescencecharacteristic of NV junctions (Fig. 2A) (Pan et al., 2000). Inthe absence of Vac8p, Nvj1p-EGFP diffuses evenly over thesurface of nuclei but is restricted to the perinuclear ER (Fig.2A) (Pan et al., 2000). As shown in Fig. 2A, Nvj1p(293-321)-EGFP failed to localize in patches corresponding to NV junctions in VAC8+ cells but, instead, appeared evenlydistributed over the nuclear envelope as in vac8- cells (Fig.2A). These data, in agreement with previous two-hybrid results(Pan et al., 2000), confirm that conserved residues at the

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immediate C-terminus of Nvj1p are necessary for bindingVac8p and creating NV junctions. This Vac8p-binding regionis not required for the strict localization of Nvj1p to the nuclearenvelope, a property that must map elsewhere in the protein.Finally, these results are consistent with our previousobservation that the C-terminal 40 residues of Nvj1p fused toGFP partially localize to the vacuole membrane in a Vac8p-dependent fashion (Pan et al., 2000).

Region I is necessary for anchoring Nvj1p to the ONMThe N-terminus of Nvj1p plays an important role in sortingto the ONM, since the addition of tags onto its N-terminuscauses the mis-localization of Nvj1p to extranuclear ER-like

Journal of Cell Science 119 (17)

membranes that form ectopic junctions with vacuoles (Kvamand Goldfarb, 2004). The first ~30 residues of the N-terminus(region I) comprise an imperfect hydrophobic sequencebordered by basic residues, reminiscent of a signal sequence(Fig. 1A,C) (Nielsen et al., 1997). Unlike signal peptidesequences, which are post-translationally cleaved, neuralnetwork algorithms predict that region I of Nvj1p is most likelynot cleaved (Bendtsen et al., 2004).

To investigate whether region I is a sorting determinant,we constructed several truncations around its hydrophobicsequence. Unlike full-length Nvj1p, which localizes strictlyto the ONM in both VAC8+ and vac8- cells (Fig. 2A), atruncated reporter lacking the hydrophobic sequence of

A

I II III IV

Nvj1p

100-75%

74-50%

aa

B

100

100

200

200

300

300

3

2

1

0

-1

-2

-3

-4

C

amino acid

hydrophobic

hydrophilic

Fig. 1. Nvj1p can be divided into four domainsbased on sequence homology across theSaccharomyces family. (A) Alignment of S.cerevisiae Nvj1p and its orthologs in closelyrelated and divergent Saccharomyces species.Amino acid sequences were obtained from theSaccharomyces Genome Database(www.yeastgenome.org). The S. kluyveriortholog of Nvj1p was identified through atblastn search of contig 02.1344 (accessionAACE02000001). Sequences were alignedusing the ClustalX program (Thompson et al.,1997). Boxes were drawn around regions of sequence similarity in the SaccharomycesNvj1p family. (B) Graphical view of residuesin Nvj1p bearing similarity or identity across

the Saccharomyces Nvj1p family. ClustalXconservation scores were calculated for eachcolumn of the alignment in 1A, and residuesbearing similarity (50-74% homology, opencircles) or near identity (75-100%, shadeddiamonds) were aligned onto a linear map of Nvj1p. These residues sorted independentlyinto four regions, as indicated. (C) Kyte-Doolittle hydropathy plot of the four conservedregions of Nvj1p. Region I at the N-terminusshows an area of considerable hydrophobicity,while region II coincides with the membrane-spanning domain of Nvj1p.

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region I (Nvj1p(1-26)-EGFP) spread into the cortical ER of vac8- cells (Fig. 2B). Similar results were observed using areporter that lacked the entire N-terminus of Nvj1p(Nvj1p(1-86)-EGFP) (Fig. 2B). These data demonstrate thatthe N-terminus is not required for ER targeting but ratherfunctions after membrane insertion. When expressed inVAC8+ cells, both truncated reporters formed atypical junctions between or along the surface of vacuolar lobes thatappeared spatially detached from the nucleus in most cells

(Fig. 2B). Thus, when bound to Vac8p, these N-terminallytruncated reporters no longer escaped to the cortical ER butrather formed extranuclear ER junctions with vacuoles.Conversely, deleting the first six residues of Nvj1p did notsignificantly affect the targeting of Nvj1p(1-6)-EGFP to theONM in vac8- cells, although a low level of cortical stainingwas sometimes visible (Fig. 2B). Moreover, Nvj1p(1-6)-EGFP formed normal perinuclear NV junctions in VAC8+ cellsrather than aberrant extranuclear junctions (Fig. 2B). Thus,residues upstream of the hydrophobic sequence in region I arenot critical for ONM sorting. In total, these experimentsdirectly implicate the hydrophobic signal anchor-likesequence of Nvj1p in proper NV-junction formation by

targeting the ONM. Interestingly, a reporter containing thefirst 90 aa of Nvj1p fused to GFP localized to mitochondria(our unpublished results), which is consistent with the fact thatregion I of Nvj1p resembles the signal-anchor sequences of the outer mitochondrial membrane proteins Tom20p andTim70p (Waizenegger et al., 2003).

The membrane-spanning domain of Nvj1p is alsorequired for ONM targeting

As described above, deleting the first 86 residues of Nvj1p didnot prevent targeting of this reporter to the ER (Fig. 2B). Sincethis deletion also retains C-terminal associations with thecytoplasmic vacuoles in VAC8+ cells, the N-terminus of Nvj1pis not required for properly orienting the protein in themembrane. Further truncation experiments revealed that afragment of Nvj1p (aa 87-120) corresponding to themembrane-spanning domain of Nvj1p (region II; Fig. 1B,C)was sufficient to localize throughout the perinuclear andcortical ER network (Fig. 2C). Thus, region II of Nvj1pcontains sufficient sequence information for targeting ERmembranes rather than the plasma membrane or otherorganelles. Importantly, an N-terminal fragment of Nvj1p

Fig. 2. Tripartite signals mediate the targeting of Nvj1pto NV junctions. (A) Region IV comprises the Vac8p-binding domain of Nvj1p. Full-length (FL) Nvj1p-EGFPand a C-terminal truncation (293-321aa) lacking the

most conserved residues of region IV were localized invac8- and VAC8+ (nvj1-) cells. Nuclear chromatin(blue) and vacuole membranes (red) were co-stainedwith Hoechst and FM4-64, respectively. Interactionsbetween Nvj1p-EGFP and Vac8p on FM4-64-stainedvacuoles create NV junctions, which appear yellow inthe overlay. The C-terminal truncation of Nvj1p doesnot form NV junctions and instead localizes uniformlyacross the nuclear envelope. (B) Region I plays a role inanchoring Nvj1p to the perinuclear ER. N-terminaltruncations preceding (1-6aa) or succeeding (1-26aa)the hydrophobic sequence of region I, or eliminating theN-terminus of Nvj1p entirely (1-86aa), were localizedin vac8- and VAC8+ (nvj1-) cells. Nuclear chromatin(blue) and vacuole membranes (red) were co-stained as

above. EGFP-tagged truncations lacking region I(denoted by a horizontal bar) escape into the cortical ERand form aberrant vacuole-associated membrane

junctions in the cytosol (see arrows) that appeardetached from the nucleus. (C) Region I and themembrane-spanning region of Nvj1p (region II)cooperatively sort to the perinuclear ER. Nvj1pfragments tagged on their C-terminus with EGFP werelocalized similarly to above. The membrane-spanningregion of Nvj1p (residues 87-120, denoted by a shadedbox) targets ER membranes but localizes exclusively tothe perinuclear ER in conjunction with region I (denotedby a horizontal bar). Replacing the transmembraneregion of Nvj1p (between residues 90-121) with the firsttransmembrane segment of Ste2p causes Nvj1p tospread into the cortical ER and form aberrant junctionswith vacuoles in the cytosol (see arrows). Numbers indiagram correspond to amino acid position.

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comprising both regions I and II (Nvj1p(121-321)-EGFP)localized strictly to the nuclear envelope (Fig. 2C). These dataillustrate that the hydrophobic N-terminal signal anchor-likesequence and the membrane-spanning domain of Nvj1p work in concert to mediate targeting to the ONM. To test whetherthe membrane-spanning domain of another protein canfunction in this regard, we replaced residues 90-120 of Nvj1p

with a slightly modified version of the first membrane-spanning domain of Ste2p, which has been used extensivelyto study co-translational insertion into the ER (see Harley andTipper, 1996). As shown in Fig. 2C, Nvj1p(Ste2pTM)-EGFPaberrantly spread into the cortical ER of vac8- cells despitethe presence of a native N-terminal domain (region I).Likewise, this chimeric reporter formed aberrant, vacuole-associated membrane junctions in VAC8+ cells (Fig. 2C),similar to those observed in cells expressing N-terminaltruncations of Nvj1p (Fig. 2B). In summary, these dataindicate that regions I and II function co-operatively to targetNvj1p into the ONM, and that the membrane-spanningdomain of Nvj1p is adapted for this purpose.

Blocking the N-terminus of Nvj1p leads to ONM-expansion and separation from the INMAs described above, region I is required for the strictlocalization of Nvj1p to the ONM. It is possible that thisdomain functions to anchor Nvj1p in the ONM by attaching tothe INM. By analogy, the ONM localization of KASH-domain-containing nesprin isoforms is mediated by interactions acrossthe perinuclear lumen with SUN domain proteins (Crisp et al.,2006). A bridging model for Nvj1p is consistent with theobservation that deleting its N-terminus results in the formationof extranuclear junctions in VAC8+ cells (Fig. 2B). Likewise,we previously reported that blocking the N-terminus of Nvj1pwith a 3HA epitope promoted the formation of analogousextranuclear junctions (Kvam and Goldfarb, 2004). If ourbridging model is true, then extranuclear ER-vacuole junctionsmight arise from sections of the ONM that have becomedetached from the INM.

To test this hypothesis we employed an N-terminally fusedGFP-Nvj1p reporter that, like N-terminal truncations (Fig. 2B),spread into the cortical ER of vac8- cells and promoted theformation of extranuclear ER junctions with vacuoles inVAC8+ cells (Fig. 3A). Anti-GFP immuno-EM was used tocharacterize the GFP-Nvj1p-labeled membranes associatedwith these extranuclear junctions. The example shown in Fig.3B shows an extension of the ONM that is sandwiched betweentwo vacuole lobes. The presence of GFP-Nvj1p in thesemembranes is confirmed by the presence of anti-GFP colloidalgold labeling (Fig. 3B, asterisks). A striking separationbetween the outer- and inner-nuclear membranes is apparentby the triangular expansion of the perinuclear lumen, which,as expected, is devoid of electron dense material such asribosomes. These results are consistent with the hypothesis thatthe N-terminus of Nvj1p mediates ONM-localization througha direct or indirect physical attachment with the INM. A yeasttwo-hybrid screen for INM proteins that might interact with theN-terminal domain of Nvj1p failed to identify potentialcandidates (our unpublished results). It is also possible that theN-terminal domain of Nvj1p inserts directly into the lipidbilayer of the INM, by analogy to the insertion of the N-terminal amphipathic helix of Sar1p into the ER (Lee et al.,


2005). Our results are also interesting because they show thatzipper-like interactions between Nvj1p and Vac8p can promotemembrane expansion in a nuclear envelope subdomain (seeCampbell et al., 2006).

Region II of Nvj1p sequesters the ER membrane proteinTsc13p

We previously demonstrated Tsc13p, an ER membrane proteininvolved in VLCFA biosynthesis, is recruited into NV junctions through associations with Nvj1p (Kvam et al., 2005).Using confocal microscopy, we mapped the region of Nvj1presponsible for interacting with Tsc13p. Briefly, N- and C-terminal truncations of Nvj1p were tested for their ability tosequester Tsc13p-EYFP from the cortical ER to the nuclearenvelope in nvj1- cells. Sequences on either side of the

Fig. 3. Blocking the N-terminus of Nvj1p promotes extranuclear junction formation by separation and extension of the ONM. (A)GFP-Nvj1p localizes throughout the perinuclear and cortical ER invac8- cells, and accumulates in extranuclear junctions between ER-like membranes and cytoplasmic vacuoles when bound to Vac8p.

Vacuole membranes (red) were stained with FM4-64 and overlayed.Images were collected by Xiaozhou Ryan. (B) Immunogoldlocalization of GFP-tagged Nvj1p by electron microscopy. GFP-tagged Nvj1p reporters were induced prior to cryofixation andimmuno-EM analysis as described in Materials and Methods. Nvj1p-GFP, a functional reporter, localizes to NV junctions. Colloidal-gold-conjugated antibodies against GFP (asterisk) label the surface of nuclei at NV junctions. By contrast, GFP-Nvj1p (a non-functionalreporter) forms extranuclear junctions with vacuoles. Here, colloidal-gold-conjugated antibodies against GFP (asterisk) label an extensionof the ONM (black arrows) that is sandwiched between two vacuolelobes. The ONM is separated from the INM (denoted by whitearrowheads), forming an extensive void in the perinuclear lumen. N,nuclei; V, vacuoles. Bars, 0.3 m.

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membrane-spanning domain (region II) of Nvj1p proveddispensable for an association with Tsc13p (Fig. 4A). Tsc13pwas efficiently sequestered to the nuclear envelope uponexpression of an N-terminal fragment of Nvj1p (aa 1-120)lacking C-terminal residues proximal to region II (Fig. 4A).Likewise, expression of a C-terminal fragment of Nvj1p (aa87-321) lacking residues preceding region II was sufficient to

sequester Tsc13p into extranuclear, vacuole-associatedmembrane junctions (Fig. 4A), which arose as a consequenceof the mis-targeting of this reporter (Fig. 2B). These datacollectively narrowed down the Tsc13p-interaction domain tothe membrane-spanning domain (region II) of Nvj1p.Alternatively, it was possible that both fragments contained

separate Tsc13p-binding activity. However, we determined thatregion II alone is sufficient to co-immunoprecipitate Tsc13p-EYFP in vivo. As shown in Fig. 4B, Tsc13p-EYFP wasefficiently co-immunoprecipitated from detergent-extractedcell lysates using a 3HA-tagged fragment of region IIcontaining the membrane-spanning domain of Nvj1p and somebordering residues (Nvj1p(87-120)-3HA). Together, these

results indicate that the sequences within region II aresufficient for binding and sequestering Tsc13p in the ER. Byanalogy, this interaction may occur between the membrane-spanning domains of these two proteins or possibly amongresidues very close to the plane of the membrane.

Fig. 4. Region II of Nvj1p sequesters Tsc13p. (A) The membrane-spanning domain of Nvj1p (region II) is responsible for recruitingTsc13p-EYFP. Full-length (FL) or N- and C-terminal truncations of

Nvj1p were overexpressed in nvj1- cells and tested for the ability tosequester Tsc13p-EYFP from cortical ER compartments to theperinuclear ER. Nuclear chromatin (blue) and vacuole membranes(red) were co-stained with Hoechst and FM4-64, respectively.Regions flanking either side of the membrane-spanning domain of Nvj1p (denoted by a shaded box) proved dispensable for thesequestration of Tsc13p-EYFP to the nuclear envelope. Numberscorrespond to amino acid positions. (B) The transmembrane regionof Nvj1p (aa 85-120) is sufficient to co-immunoprecipitate Tsc13p-EYFP in vivo. Detergent-extracted lysates of cells expressingTsc13p-EYFP and 3HA-tagged Nvj1p(85-120aa) or empty vectorwere prepared as described in Materials and Methods. Nvj1p(85-120aa)-3HA was immunoprecipitated (IP) with anti-HA conjugatedagarose beads.

Fig. 5. Region III of Nvj1p is necessary and sufficient to bind Osh1p.(A) Deletion of the most conserved residues in region III abrogatesthe Nvj1p-dependent sequestration of GFP-Osh1p to the nuclearenvelope. Full-length (FL) or N- and C-terminal truncations of Nvj1pwere overexpressed in nvj1- cells and tested for the ability tosequester GFP-Osh1p from soluble and Golgi-associated pools to thenuclear surface. Conserved residues in region III of Nvj1p (aa 130-176) proved necessary for GFP-Osh1p sequestration. Numberscorrespond to amino acid positions. (B) Residues 130-177 of Nvj1pare sufficient to co-immunoprecipitate GFP-Osh1p in vivo. Lysatesof cells expressing GFP-Osh1p and 3HA-tagged Nvj1p(130-177aa)or empty vector were prepared as described in Materials andMethods. Nvj1p(130-177aa)-3HA was immunoprecipitated (IP) withanti-HA conjugated agarose beads.

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Region III is sufficient and necessary to bind Osh1pWe previously showed that Nvj1p sequesters Osh1p into NV junctions in a Vac8p-independent fashion, most likely by directbinding (Kvam and Goldfarb, 2004). In order to identify theregion of Nvj1p responsible for interacting with Osh1p, weexpressed N- and C-terminal truncations of Nvj1p in nvj1-cells and assessed their ability to recruit GFP-Osh1p from

soluble and Golgi-associated pools to the nuclear envelope byconfocal microscopy. As shown in Fig. 5A, the putative Osh1p-binding site mapped to a segment of Nvj1p (aa 120-177)adjacent to the membrane-spanning domain. Importantly, thisarea coincided with region III of Nvj1p (Fig. 1A,B) and, likethe Vac8p-binding domain (region IV), is exposed to thecytoplasm. Saccharomyces orthologs of Nvj1p contain anespecially well conserved sequence between residues 140-174in region III (Fig. 1A,B). We created a myc-tagged Nvj1pmutant with an internal deletion that eliminated theseconserved residues. Expression of this mutant, Nvj1p(130-176)-myc, was confirmed by immunoblot (our unpublisheddata). As expected, overexpression of Nvj1p(130-176)-mycfailed to sequester GFP-Osh1p from surrounding cytoplasmic

and Golgi compartments (Fig. 5A). This fact was not due toimproper targeting of the Nvj1p(130-176)-myc reporter, sincea version tagged with GFP localized exclusively to the ONM,where it formed normal NV junctions in VAC8+ cells (our


unpublished data). Moreover, expression of Nvj1p(130-176)-myc was sufficient to sequester Tsc13p-EYFP from the corticalER to NV junctions (our unpublished data). In biochemicalpull-down experiments, GFP-Osh1p efficiently co-immunoprecipitated with an epitope-tagged fragment of regionIII encompassing residues 130-177 of Nvj1p (Nvj1p(130-177)-3HA) (Fig. 5B). Together, these results show that aconserved sequence within region III is both necessary andsufficient for an interaction between Nvj1p and Osh1p.

NVJ1-overexpressing cells exhibit a cold-sensitivetryptophan transport defect related to Osh1pSeveral studies have demonstrated that deleting OSH1 intryptophan auxotrophs (trp1) produces a temperature-sensitivecell growth defect on media containing low concentrations of tryptophan (Loewen et al., 2003; Levine and Munro, 2001;Jiang et al., 1994). This growth defect is similar to that of erg6- cells, which are defective in ergosterol biosynthesis, and islikely related to the sterol-dependent sorting of the high-affinity tryptophan permease Tat2p to the plasma membrane(Umebayashi and Nakano, 2003; Gaber et al., 1989). This

sterol-sensitive step occurs during the sorting of Tat2p intodetergent-resistant membrane domains (DRMs, or lipid rafts)within late Golgi or post-Golgi compartments (Umebayashiand Nakano, 2003; Gaber et al., 1989). Based on these results,

we reasoned that the overexpression of Nvj1p mightcause an analogous slow-growth phenotype bysequestering Osh1p into NV junctions and away from thetrans-Golgi. Indeed, compared to control cells expressingan empty vector, NVJ1-overexpressing cells werehypersensitive to tryptophan limitation at lowtemperature (Fig. 6A). This growth defect wascomplemented by excess tryptophan or highertemperature (Fig. 6A), analogous to what was observedin osh1- cells (Levine and Munro, 2001). Moreover, thegrowth defect of NVJ1-expressing cells was rescued bysingle copy CEN plasmids expressing either TAT1 orTAT2 tryptophan permeases under the control of their

Fig. 6. Overexpression of Nvj1p, but not a mutant formlacking Osh1p-binding activity, confers a tryptophantransport-related growth defect. (A) Cells overexpressingNvj1p, but not a mutant form lacking the Osh1p-bindingdomain, grow poorly on media containing limitingconcentrations of tryptophan at low temperature. Cellsharboring PCUP1-NVJ1, PCUP1-NVJ1(130-176aa)-myc , orempty vector were induced and replica-plated onto mediacontaining limiting (15 g/ml) or excess (40 g/ml)

tryptophan as described in Materials and Methods. (B) Thecold-sensitive growth phenotype of Nvj1p-overexpressingcells is suppressed by low- and high-affinity tryptophanpermeases. Cells harboring PCUP1-NVJ1 and either pTAT1,pTAT2, or empty vector were induced and replica-plated ontomedia containing limiting (15 g/ml) or excess (40 g/ml)tryptophan as above. (C) NV junctions are not necessary toconfer tryptophan transport defects in Nvj1p-overexpressingcells. Growth curves of vac8- cells expressing PCUP1-NVJ1(open squares), PCUP1-NVJ1(130-176aa)-myc (black triangles), or empty vector (gray circles) were determined inliquid media containing limiting (15 g/ml) or excess (40g/ml) tryptophan at 25°C using a Bioscreen C Analyzer (seeMaterials and Methods).

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native promoters (Fig. 6B). By contrast, cells overexpressingNvj1p(130-176)-myc, which lacks Osh1p-binding activity(Fig. 5A), showed normal growth on low tryptophan media(Fig. 6A). These results were corroborated by monitoring cellgrowth rates at 25°C in liquid media containing low or highconcentrations of tryptophan (Fig. 6C). The ability of Nvj1p toform NV junctions proved to be unimportant for these

tryptophan transport-related growth phenotypes, since similarresults were observed in vac8- cells (Fig. 6C). In summary,these results demonstrate that the sequestration of Osh1p byNvj1p causes a defect in tryptophan uptake similar to thatpreviously reported for osh1- cells.

NVJ1+ trp1 cells grow poorer in tryptophan-depletedmedia than nvj1- trp1 cellsOsh1p localizes to both trans-Golgi membranes and NV junctions, but is found almost exclusively at NV junctions asNvj1p levels rise by ectopic overexpression or as aconsequence of nutrient depletion during late-log phase (Kvamand Goldfarb, 2004; Levine and Munro, 2001). Because highlevels of Nvj1p affect cell growth by sequestering Osh1p (Fig.

6), we were interested in determining whether physiologicallyrelevant levels of Nvj1p were active in this regard. For thispurpose, we analyzed the growth of isogenic NVJ1+ trp1 andnvj1- trp1 cells as a function of tryptophan limitation. At

concentrations of tryptophan sufficient to slow the growth of NVJ1-overexpressing cells (15 g/ml), the growth of NVJ1+

and nvj1- cells at 25°C was indistinguishable (Fig. 7B).However, nvj1- cells grew markedly better than NVJ1+ cellswhen the concentration of extracellular tryptophan becameincreasingly limiting (5 g/ml) (Fig. 7A). This difference,albeit small, is significant and was consistently reproduced in

several experimental trials. Thus, native levels of Nvj1p maybe mildly deleterious for growth in tryptophan-depletedenvironments by affecting the localization and activity of Osh1p.

DiscussionNvj1p is a remarkable protein that mediates several novelphenomena, including the formation of the only known inter-organelle membrane junction apparatus (NV junctions), and aunique type of autophagy that pinches-off and degrades non-essential parts of the yeast nucleus (PMN). The key findingsof this study include mapping the regions of Nvj1p responsiblefor sorting to the ONM and interacting with its binding-partners, Vac8p, Osh1p and Tsc13p. These proteins bind, either

directly or indirectly, to discrete, non-overlapping regions of Nvj1p. The functional domains identified in this study map tofour regions (I-IV) of Nvj1p that are conserved among theSaccharomyces family of NVJ1 orthologs. Some of thesesequence regions are present in increasingly divergent Nvj1p-like proteins in more evolutionarily distant yeasts, includingKluveromyces waltii, Kluveromyces lactis, Candida glabrataand Ashbya gossypii (not shown). It should be noted that thegenomes of all fungal species whose genomes have beensequenced, including Aspergillis nidulans and Neurosporacrassa, encode well-conserved orthologs of VAC8, TSC13 andOSH1. Our results also reveal a novel role for Nvj1p in theregulation of tryptophan transport. These experiments are thefirst to expose a growth defect associated with NVJ1expression. From our results, we propose that the sequestrationof Osh1p by Nvj1p inhibits tryptophan uptake from theenvironment, possibly by modulating tryptophan permease-trafficking to the plasma membrane via a mechanism that waspreviously shown to require Osh1p (Levine and Munro, 2001;Jiang et al., 1994).

The sorting of eukaryotic proteins to the ONM is usuallyexplained in terms of binding to resident proteins of the nuclearenvelope, which begs the question of how the resident proteinsthemselves become localized. We show that the N-terminalhydrophobic domain (region I) and the membrane-spanningdomain (region II) of Nvj1p are both required for efficientlocalization to the ONM. Region I contains a signal anchor-like sequence consisting of a relatively short, imperfecthydrophobic sequence flanked by positively charged residues.This sequence is not required for either the ER-targeting of Nvj1p or its correct orientation in the membrane, since N-terminal truncations of Nvj1p retain C-terminal associationswith both Osh1p and Vac8p in the cytoplasm. The cytoplasmicC-terminal domain of Nvj1p (aa 121-321) is dispensable forONM sorting, which rules out a role for Vac8p or Osh1p inthis regard. However, Nvj1p-truncations lacking region I failto localize strictly to the ONM and, instead, are foundubiquitously throughout the perinuclear and cortical ER. Themembrane-spanning domain within region II plays anadditional role in sorting to the ONM and, as discussed below,

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Fig. 7. Native levels of Nvj1p are mildly deleterious for growth intryptophan-depleted media. nvj1- and its isogenic parental ( NVJ1+)tryptophan auxotrophic strain (trp1) were grown in rich media to log-phase and shifted into SC media containing limiting (15 g/ml) ordepleted (5 g/ml) concentrations of tryptophan. Cell growth wasmonitored at 25°C using a Bioscreen C Analyzer (see Materials andMethods). Error bars were calculated from two independent trialsperformed in duplicate.

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also interacts with Tsc13p. A 47-residue segment of region IIis sufficient to target a GFP reporter to the ER network. Wefound that the important function of this domain in ONMsorting cannot be substituted by the first membrane-spanningdomain of Ste2p, despite the fact that this chimera wasefficiently expressed in the correct orientation in the ER. Thus,these results demonstrate that both regions I and II contribute

specific information for ONM sorting.NV-junction formation requires direct interactions between

Nvj1p in the ONM and Vac8p on the vacuole membrane;however, the sorting of Nvj1p to the ONM is not dependent onVac8p (Pan et al., 2000). We mapped the Vac8p-bindingdomain of Nvj1p to its C-terminus (region IV), which isconsistent with previous two-hybrid results (Pan et al., 2000).When the sorting of Nvj1p is altered (either by blocking ormutating region I or through substitution of region II), Nvj1pspreads into the cortical ER and forms aberrant junctions withVac8p-associated vacuole membranes. As shown in Fig. 3,these extranuclear junctions originate as extensions of theONM. The N-terminal signal anchor-like sequence of Nvj1p,together with the membrane-spanning domain, may mediate a

physical interaction across the perinuclear lumen with the INM(Fig. 8). Such a bridge would anchor Nvj1p in the ONMand prevent its escape into intermediate and cortical ERcompartments. A physical connection to the INM would alsoexplain how the ONM and INM move in concert into vacuoleinvaginations during PMN (Roberts et al., 2003). In support of this notion, blocking the N-terminus of Nvj1p with GFPpromotes the separation of the inner and outer nuclearmembranes, leading to expansion of the ONM throughmultiple zipper-like interactions between Nvj1p and Vac8p onthe vacuole surface. Precedent for a nuclear envelope bridgingapparatus comes from recent reports describing the LINCcomplex, which involves interactions between KASH-domain-containing nesprin isoforms in the ONM with SUN domainproteins in the INM (reviewed in Worman and Gundersen,2006; Starr and Fischer, 2005). The ONM localization of nesprin requires that it be anchored to a SUN domain proteinin the INM, since the depletion or the secretion of SUNdomains into the ER lumen results in the mislocalization of nesprin to the bulk ER (Crisp et al., 2006).

In addition to mediating ONM localization, region II of Nvj1p also functions in sequestering Tsc13p within the ER.Our results are consistent with a model in which themembrane-spanning domain of Nvj1p interacts with one ormore of the membrane-spanning domains of Tsc13p within theER bilayer. Associations among membrane-spanning domains


are known to stabilize certain integral membrane proteincomplexes (Li et al., 2003). Interactions between Nvj1p andTsc13p, which are likely to be direct, may occur within thehydrophobic interior of the membrane or via residuesimmediately flanking their membrane-spanning segments. Inthis regard, it is interesting that the co-localization of Tsc13pwith Nvj1p at NV junctions may depend on the availability of

substrate long chain fatty acids (Kvam et al., 2005).Finally, this report reveals that a conserved sequence in

region III of Nvj1p is necessary and sufficient to bind Osh1p.The sequestration of Osh1p into NV junctions, first shown byLevine and Munro (Levine and Munro, 2001), is mediated byVac8p-independent interactions with Nvj1p (Kvam andGoldfarb, 2004). Osh1p is particularly interesting because of its likely role in trafficking the high-affinity tryptophanpermease, Tat2p. Tat2p is a constitutively expressed membranepermease that is routed to either the plasma membrane orvacuole depending on the concentration of tryptophan in themedium (Beck et al., 1999). A recent study revealed that Tat2ptrafficking is dependent on its association with detergent-resistant membrane domains (DRMs) in post-Golgi

compartments (Umebayashi and Nakano, 2003). In fact,several mutations disrupting the synthesis of DRM-associatedlipids confer pleiotropic effects on tryptophan transport,including those affecting the synthesis of ergosterol (erg6-,erg3-, kes1-), sphingolipids (elo2-), GPI-anchor formation(gwt1-10), and specific phospholipids (cho1-) (Okamoto etal., 2006; Umebayashi and Nakano, 2003; Chung et al., 2001;Nakamura et al., 2000; Hemmi et al., 1995; Jiang et al., 1994).Likewise, cells lacking Osh1p are defective in [3H]tryptophanuptake and osh1- trp1 cells exhibit a severe growth defect onlow tryptophan media (Levine and Munro, 2001; Jiang et al.,1994). Taken together, these published results stronglyimplicate Osh1p as a key player in the sterol-dependenttrafficking of Tat2p to the plasma membrane.

Here, we report that overexpression of Nvj1p in trp1auxotrophs causes a slow-growth phenotype similar to thatreported for osh1- cells (Levine and Munro, 2001). Thisgrowth defect is dependent on the sequestration of Osh1p,since cells overexpressing a mutant version of Nvj1p withoutan Osh1p-binding domain (region III) grew normally. TheNvj1p-dependent sequestration of Osh1p may provide a readymechanism to downregulate tryptophan permease activity inresponse to nutrient depletion. In support of this notion, nativelevels of Nvj1p confer a moderating effect on cell growth inlow-tryptophan media, presumably due to the steady (butphysiologically significant) sequestration of Osh1p. As cells

ivacuole membrane

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Fig. 8. Molecular model of NV junctions. Nvj1p is restricted tothe outer nuclear membrane, which is continuous with theperinuclear ER. The membrane-spanning domain (II) of Nvj1pinteracts with Tsc13p in the ER membrane. An adjacentcytosolic region (III) associates with Osh1p. The C-terminaltail of Nvj1p (IV) binds to Vac8p, which is acylated to thevacuole membrane. The hydrophobic N-terminus of Nvj1p (I)may link the inner- and outer-nuclear membranes either bybinding an unknown factor (X) in the INM (panel i), or byinserting directly into the INM by spanning across theperinuclear lumen (panel ii).

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progress into late log phase, GFP-Osh1p becomes increasinglyassociated with NV junctions and less so at trans-Golgicompartments (Levine and Munro, 2001) (our unpublishedresults). It is during late log phase when high-affinity nutrientpermeases such as Tat2p are routed to the vacuole and degraded(Schmidt et al., 1998). These observations are consistent withthe hypothesis that Nvj1p is a negative regulator of Osh1p

function and, as such, suggest that Nvj1p plays a novel role insterol-dependent protein trafficking. Previous studies have alsoimplicated the yeast homolog of mammalian VAMP-associatedprotein, Scs2p, in targeting Osh1p and other yeast ORPs tocellular membranes, although Scs2p is not strictly required forthe localization of GFP-Osh1p to NV junctions (Loewen et al.,2003). Further work will be necessary to tease-out the roles of Nvj1p, Scs2p and other cellular factors in the complex controlOsh1p function.

The faster growth of nvj1- trp1 cells in limiting tryptophansuggests that Nvj1p may normally function as a finely tunedrheostat to control Osh1p activity, even during log phase whenNvj1p levels are normally low. Our results may also accountfor the increased survival of nvj1- cells during stationaryphase, which is a function of chronological aging (see Fabrizioet al., 2005). A recent genome-wide screen identified mutantsin NVJ1 and a number of genes implicated in nutrient sensingand TOR signaling as some of the longest-lived in the deletioncollection (Powers et al., 2006). Perhaps not coincidentally,TOR signaling has also been implicated in the degradation of high-affinity nutrient transporters (including Tat2p) in favor of lower-affinity transporters during nutrient stress (Edinger andThompson, 2002; Beck et al., 1999; Schmidt et al., 1998).

In conclusion, we have mapped several functional domainsof Nvj1p that mediate the recruitment of Vac8p, Osh1p andTsc13p to the ONM, and revealed roles for both the N-terminalsignal anchor-like sequence and membrane-spanning domainof Nvj1p in sorting to the ONM. Finally, we describe afunctional link between the sequestration of Osh1p into NV junctions and the regulation of sterol-dependent proteintrafficking to the plasma membrane. This mechanism may havebroader implications for the control of ORP function in highercells.

Materials and MethodsYeast strains and growth conditionsYeast strains used in this study were based in the YEF473 genetic background (trp1-

63 leu2-1 ura3-52 his3-200 lys2-801) (Bi and Pringle, 1996). Deletion of NVJ1and VAC8 in YEF473 was described elsewhere (Pan et al., 2000; Pan and Goldfarb,1998). Unless otherwise indicated, cells were cultured at 30°C in YPD, syntheticcomplete media (SC), or standard dropout media containing 2% glucose (Sherman,1991).

PlasmidsPlasmids for the expression of NVJ1, NVJ1-GFP or GFP-NVJ1 under CUP1promoter control (PCUP1- NVJ1, PCUP1- NVJ1-GFP, PCUP1-GFP- NVJ1) weredescribed previously (Pan et al., 2000). Nvj1p truncations were constructed by PCR-amplifying fragments of NVJ1 corresponding to the indicated amino acid positionswith primers that introduced EcoRI and HindIII sites. These fragments were ligatedinto the EcoRI and HindIII sites of pEGFP-C-FUS, which was constructed frompGFP-C-FUS (Niedenthal et al., 1996) by replacing GFP with a PCR-amplifiedClaI- EGFP-SalI fragment generated from pEGFP (Clontech, CA). To express non-fluorescent versions of these truncations, NVJ1 fragments were subcloned frompEGFP-C-FUS into the PGAL1/10 plasmid pESC(HIS) (Stratagene) using EcoRI andClaI sites. For immunoprecipitation experiments, the FLAG epitope of pESC(HIS)was replaced with the triple HA epitope from pGTEPI (Tyers et al., 1992) usingSpeI and SacI sites, and PCR-amplified fragments of NVJ1 were cloned upstreamof 3HA using EcoRI and SpeI sites. Deletion of residues 130-176 of Nvj1p wasaccomplished by fusing two PCR-amplified fragments of NVJ1, corresponding to

residues 1-129 and 177-321-myc of PGAL1- NVJ1-myc (Kvam and Goldfarb, 2004),into the multiple cloning site of the PCUP1 plasmid, pRK2 (Pan et al., 2000). Toreplace the membrane-spanning region of Nvj1p with that of Ste2p, an XholI-substituted chimeric NVJ1 / STE2 reverse primer (5-ccgctcgaggacaatcaaagtcaa-agcagctgcaccacatatgacaccaaacataatggccaattcgctggattgtctgg-3), corresponding toresidues 84-90 of Nvj1p and residues 52-68 of Ste2p [including the mutation R58Iused by Harley and Tipper (Harley and Tipper, 1996)], was used to PCR-amplify afragment corresponding to the first 90 residues of NVJ1. This chimeric fragmentwas ligated to a second NVJ1 PCR fragment (corresponding to residues 121-321)

using introduced XholI sites, and cloned into pEGFP-C-fus. Plasmids expressingchimeric or truncated version of NVJ1 were confirmed by sequencing. Plasmids forexpression of the tryptophan permease genes TAT1 and TAT2 (pTAT1 and pTAT2)were generously supplied by M. Hall (Schmidt et al., 1994). Plasmids for theconstitutive expression of GFP-Osh1p (pRS416-GFP-OSH1) or the inducibleexpression of Tsc13p-EYFP (PCUP1-TSC13- EYFP) were also described previously(Kvam et al., 2005; Levine and Munro, 2001).

Growth assays for Nvj1p-overexpressing and nvj1- cellsGrowth sensitivity on solid media was assayed using previously described methodsfor tryptophan uptake (Levine and Munro, 2001). Briefly, log-phase trp1 cellsharboring the indicated PCUP1 plasmids were induced for 3 hours with 0.1 mMCuSO4. Three OD600 units of culture were then collected by centrifugation. Cellpellets were resuspended in 1 ml of fresh media containing 0.1 mM CuSO4, dilutedserially by tenfold, and plated onto selective media containing limiting (15 g/ml)or excess (40g/ml) concentrations of tryptophan using a multi-pronged replicator.Plates were incubated at 25°C or 37°C for 72-96 hours. For growth-curve analyses

in liquid media, log-phasetrp1

cells harboring the indicated PCUP1 plasmids wereinduced for 3 hours with 0.1 mM CuSO4. A total of 0.25 OD600 units of culturewere collected by centrifugation and inoculated into 5 ml of SC media containinglimiting (15 g/ml) or excess (40 g/ml) tryptophan and 0.1 mM CuSO4.Approximately 350 l of culture were pipetted into triplicate wells of aHoneycomb 2 cuvette multiwell plate (MTX Lab Systems, Vienna, VA). Cells weregrown at 25°C in a Bioscreen C Analyzer (MTX Lab Systems) programmed tomeasure the optical density (600 nm) of the Honeycomb 2 plate every 20 minutes,with medium shaking for 10 seconds prior to each reading. Data points werecollected using EZExperiment software (MTX Lab Systems). Triplicate readingswere averaged for each strain. A similar protocol was used to measure the growthof nvj1- and its parental trp1 strain in SC media containing 5 g/ml or 15 g/mlof tryptophan.

Immunoprecipitation and immunoblottingCells harboring PGAL1/10- NVJ1(130-177aa)-3HA, PGAL1/10- NVJ1(85-120aa)-3HA,or empty vector and co-expressing either pRS426-GFP-OSH1 or PCUP1-TSC13-

EYFP were cultured in selective media containing 2% raffinose to log-phase.

Protein expression was induced with 2% galactose or, where appropriate, 0.1 mMCuSO4 for 3.5 hours. Approximately 50 OD600 units of culture were collected bycentrifugation. Cell pellets were weighed and overlayed with an equivalent volumeof acid-washed 425-600 m glass beads (Sigma) and 2 volumes of extractionbuffer (EB) (40 mM Tris-HCl pH 7.5, 100 mM NaCl, 2 mM DTT, 0.5 mM EDTA)containing CompleteTM protease inhibitors (Roche, Mannheim, Germany) and 2g/ml pepstatin A, aprotinin, and leupeptin (Sigma). Cells were vortexed five timesfor 2 minutes, intermittent with 2 minute incubations on ice. Lysate suspensionswere solubilized with 1% NP-40, vortexed, and cleared by centrifugation (500 g

for 10 minutes). After collecting the supernatant, glass beads were washed with 2volumes of EB, vortexed, and cleared by centrifugation. Combined supernatantswere incubated with 30 l of goat IgG-agarose for 15 minutes with rotation toremove non-specific peptides. After brief centrifugation, the total proteinconcentration of the cleared lysate was determined by Bradford assay.Approximately 3 mg of total protein was transferred into MicroSpin Columns(Amersham) and the final volume was brought up to 0.5 ml with EB whennecessary. Lysates were incubated with 30 l of goat anti-HA conjugated agaroseat 4°C overnight with rotation (Santa Cruz Biotechnology). Bead complexes were

isolated by centrifugation (500 g for 1 minute), washed four times with HNTG (20mM HEPES pH 7.5, 0.15 M NaCl, 0.1% Triton X-100, 10% glycerol), and elutedinto a fresh tube with 35 l of 2 protein gel sample loading buffer (100 mM Tris-HCl pH 6.8, 2% SDS, 20% glycerol, 2% 2-mercaptoethanol, 0.1% bromophenolblue). Eluates were boiled for 5 minutes, and 10-15 l of sample were analyzedby 8% and 18% SDS-PAGE. GFP-Osh1p and Tsc13p-EYFP were probed byImmuno blot with polyclonal BD Living ColorsTM A.v. peptide antibodies(Clontech). HA-tagged Nvj1p fragments were probed with mouse anti-HAmonoclonal antibodies (Santa Cruz Biotechnology). All immuno-probed proteinswere detected using alkaline phosphatase-coupled goat anti-rabbit IgG (Santa CruzBiotechnology) or alkaline phosphatase-coupled goat anti-mouse IgG (Zymed) anddeveloped colometrically

Cell imaging and confocal microscopyUnless otherwise indicated, cells were induced for specific reporters at log-phase.EGFP-labeled truncations of Nvj1p were expressed from pEGFP-C-FUS by

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culturing in media lacking methionine for 3 hours. Non-fluorescent truncations of

Nvj1p were expressed from pESC(HIS) by shifting cells from raffinose- to

galactose-containing media and culturing for 3 hours. Where indicated, Tsc13p-

EYFP was co-expressed for 3 hours upon addition of 0.1 mM CuSO4 to the media.

Vacuoles were stained with FM4-64 as described previously (Pan and Goldfarb,

1998). Nuclear chromatin was stained immediately prior to microscopic analysis

with 5 M Hoechst reagent H-1398 (Molecular Probes). Confocal microscopy wasperformed on a Leica TCS NT microscope equipped with a 100X Fluorotar lens

and UV, Ar, Kr/Ar, and He/Ne lasers (Leica Microsystems, Chantilly VA). Images

were processed using Adobe PhotoShop 5.0 (Adobe Systems, CA). Panels reflect

the localization phenotypes observed in all cells overexpressing the indicated

reporters.

Immunoelectron microscopyWild-type cells harboring PCUP1- NVJ1-GFP or PCUP1-GFP- NVJ1 were grown to logphase and induced for 1 hour with 0.1 mM CuSO4. Cells were high-pressure frozen,

freeze substituted, sectioned, and stained as previously described (Giddings et al.,

2001). GFP-tagged Nvj1p was detected by immuno-EM using affinity purified

rabbit polyclonal anti-GFP antibody and gold-conjugated anti-rabbit secondary

antibody. Serial thin sections were viewed with a CM10 electron microscope

(Philips Electronic Instruments, Mahwah, NJ) and images were captured with a

GATAN digital camera.

We thank T. H. Giddings and J. B. Meehl for their expert electronmicroscopy, and Xiaozhou Ryan for the confocal images in Fig. 3.

We also thank Rebecca Gilson for assistance with tryptophan uptakeassays, Michael Hall for providing pTAT1 and pTAT2 plasmids, TimLevine for providing pRS416-GFP-OSH , and Bill Burke for helpfulcomments on the manuscript. This study was supported by USNational Institutes of Health RO1 grant GM67838 (to D.S.G.).

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