3622

Research Article

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: dasg@mail.rochester.edu)

Journal of Cell Science

Accepted 9 June 2006

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

Summary
Nvj1p resides in the outer nuclear membrane (ONM) and
binds the vacuole membrane protein Vac8p to form
nucleus-vacuole (NV) junctions in Saccharomyces
cerevisiae. The induction of NVJ1 expression during
starvation results in the sequestration of two additional
binding partners, Tsc13p and Osh1p. Here, we map the
domains of Nvj1p responsible for ONM targeting and
partner binding. ONM targeting requires both the Nterminal signal anchor-like sequence and the topogenic
membrane-spanning domain of Nvj1p. The N-terminal
signal anchor-like sequence may anchor Nvj1p in the ONM
by bridging to the inner nuclear membrane. A region
encompassing the membrane-spanning domain is sufficient
to bind Tsc13p. Osh1p and Vac8p bind to distinct regions
in the cytoplasmic tail of Nvj1p. Overexpression of Nvj1p

Key words: Nuclear envelope, Endoplasmic reticulum, Nucleusvacuole junctions, Tryptophan permease, Osh1p, Tsc13p, Nvj1p

Introduction
The endoplasmic reticulum (ER) is an interconnected
membrane network composed of structurally distinct
compartments, including the peripheral or cortical ER and the
perinuclear ER, which surrounds the nucleus (reviewed in
Voeltz et al., 2002). Although these compartments are
topologically continuous and share a common lumen, each has
unique properties and functions (reviewed in Levine and
Rabouille, 2005; Voeltz et al., 2002). The perinuclear ER,
which comprises the nuclear envelope (NE), consists of two
concentric membrane sheets connected at fenestrae called
nuclear pores. The inner nuclear membrane (INM) serves as
an attachment sites for chromatin and, in higher eukaryotes, is
associated 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, contains
cytoskeletal attachment sites for nuclear positioning (reviewed
in Hetzer et al., 2005). The mechanisms responsible for
creating and maintaining these structurally distinct
compartments are poorly understood.
Accurate protein sorting to the nuclear envelope is critically
important for nuclear function and cell survival. Mutations
affecting the targeting and function of specific nuclear
envelope proteins have been linked to several human diseases
collectively termed nuclear envelopathies (reviewed in Somech
et al., 2005). In general, proteins localize to the nuclear
envelope through associations with resident nuclear proteins

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

occurs initially via energy-dependent interactions with the
nuclear pore complex (NPC) (Ohba et al., 2004; Beilharz et
al., 2003). Nuclear lamins exemplify an important class of
peripheral INM proteins that are accompanied across the NPC
by soluble receptors called karyopherins (reviewed in Hetzer
et al., 2005). Likewise, tail-anchored INM proteins translocate
through the NPC and insert post-translationally into the INM
(Beilharz et al., 2003). However, integral INM proteins that are
synthesized in the extranuclear rough ER are sorted to the INM
probably by diffusion across the continuous membrane of the
nuclear pore (Ohba et al., 2004). Saturable interactions with
nuclear lamins, chromatin, or other nuclear proteins are
thought to retain these proteins within the INM (Ostlund et al.,
2006).
Mechanisms governing the sorting of ONM proteins are
poorly characterized, as only a few cases are known. A WPP
domain (tryptophan-proline-proline) at the N-terminus of a
subset of plant proteins is necessary and sufficient to target the
ONM, presumably by interacting with factors on the nuclear
surface (Patel et al., 2004). Additionally, metazoan KASHdomain-containing proteins (Klarsicht, ANC-1, and Syne
homology) are retained in the ONM through interactions across
the perinuclear lumen with specific INM proteins (reviewed in
Worman 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

in trp1 cells causes a growth defect in low tryptophan that

is rescued by additional copies of TAT1 or TAT2 tryptophan
trp1 cells grow faster than
permeases. Conversely, nvj1-
NVJ1+ trp1 cells in limiting tryptophan. Importantly,
deleting the Osh1p-binding domain of Nvj1p abrogates the
tryptophan transport-related growth defect of Nvj1poverexpressing cells. Therefore, the Nvj1p-dependent
sequestration of Osh1p negatively regulates tryptophan
uptake from the medium, possible by affecting the
trafficking of tryptophan permeases to the plasma
membrane.

Journal of Cell Science

Functional domains of Nvj1p

anchored to the INM by nuclear lamins (Crisp et al., 2006).
Together, KASH and SUN proteins mediate the assembly
of large nuclear envelope-associated complexes (LINC
complexes) that physically link the cytoskeleton to
nucleoplasmic components.
By analogy to KASH proteins, Nvj1p is a yeast membrane
protein 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, Nvj1p
localizes evenly over the surface of the nuclear envelope but
fails to escape into the cortical ER, even when overexpressed
(Pan et al., 2000). Thus, an undetermined mechanism mediates
the strict localization of Nvj1p to the ONM. Aside from Vac8p,
Nvj1p also sequesters two proteins with roles in lipid
homeostasis, Osh1p and Tsc13p, to the nuclear surface (Kvam
et al., 2005; Kvam and Goldfarb, 2004). The fraction of Osh1p
or Tsc13p present at NV junctions depends on the expression
level of Nvj1p, which is upregulated in response to nutrient
depletion. Tsc13p is an essential ER membrane protein
involved in the synthesis of very-long-chain fatty acids
(VLCFAs), which are important constituents of ceramides,
sphingolipids, GPI anchors and unusual inositolphospholipids
and, as such, play important roles in membrane fluidity, lipid
raft biogenesis, and cell signaling (Eisenkolb et al., 2002;
Kohlwein et al., 2001; Dickson, 1998). Osh1p, on the other
hand, is a member of a large family of eukaryotic oxysterolbinding protein-related proteins (ORPs) that share a conserved
oxysterol-binding domain, which forms a hydrophobic sterolsensing cavity (Im et al., 2005). Yeast lacking the overlapping
Osh protein family (Osh1p-Osh7p) show significant defects in
sterol-dependent processes including endocytosis, vesicle
transport, and homotypic vacuole fusion (Beh and Rine, 2004).
Consistent with a role in sterol regulation, osh1- cells exhibit
a cold-sensitive tryptophan transport defect akin to erg6
mutants that are defective in ergosterol synthesis (Levine and
Munro, 2001; Jiang et al., 1994). Ergosterol is required for
trafficking of the high-affinity tryptophan permease, Tat2p,
to the plasma membrane via detergent-resistant membrane
domains (Umebayashi and Nakano, 2003). The localization of
Osh1p is regulated by several targeting determinants, including
a PH domain specific for Golgi membranes (Levine and
Munro, 2001), a FFAT motif (FF in an acidic tract) that targets
the ER through associations with Scs2p (Loewen et al., 2003),
and an N-terminal ankyrin repeat domain that mediates
localization to NV junctions (Levine and Munro, 2001).
NV junctions are sites of a starvation-induced autophagic
process called piecemeal microautophagy of the nucleus
(PMN) that degrades portions of the yeast nucleus in the
hydrolytic vacuole lumen (Roberts et al., 2003). During PMN,
the vacuole membrane envelopes a section of the nuclear
envelope through invagination, forming a nuclear bleb.
Ultimately, this tri-laminar PMN bleb, consisting of the innerand outer-nuclear membranes and the vacuole membrane, is
pinched-off as a vesicle and degraded in the vacuole lumen
(Roberts et al., 2003). PMN appears to respond to nutrient
starvation by scavenging and recycling nonessential nuclear
components, including portions of the nuclear envelope and,
often, underlying nucleolar pre-ribosomes (Roberts et al.,
2003). Activities associated with Tsc13p and the Osh-protein
family are required for efficient PMN biogenesis (Kvam et al.,
2005; Kvam and Goldfarb, 2004).

3623

In this study, we map the sequences of Nvj1p that mediate

sorting to the ONM and associations with Vac8p, Tsc13p and
Osh1p. The N-terminal signal anchor-like sequence of Nvj1p is
not required for either targeting to the ER or correct orientation
in the membrane, but is required for ONM anchoring, possibly
by 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 in
the cytoplasmic C-terminus of Nvj1p. Based on growth
phenotypes associated with the Osh1p-Nvj1p interaction, we
present evidence that Nvj1p functions as a negative regulator of
tryptophan transport during nutrient depletion.
Results
Saccharomyces orthologs of Nvj1p share four
conserved domains
Nvj1p is an integral ER membrane protein that localizes
exclusively to the ONM and sequesters proteins from diverse
cellular pools to the nuclear surface. Since Nvj1p lacks known
sequence motifs, we identified functionally constrained regions
in Nvj1p by comparing orthologous protein sequences from six
Saccharomyces species, including four stricto senso species
similar to S. cerevisiae (S. paradoxus, S. mikatae, S. bayanus
and S. kudriavzevii) and two more divergent species (S.
castellii and S. kluyveri) (Cliften et al., 2001). These
alignments revealed four conserved regions in Nvj1p (Fig.
1A,B). Region I at the N-terminus is characterized by a
hydrophobic signal anchor-like sequence (Fig. 1B,C). Region
II overlaps with the predicted membrane-spanning domain of
Nvj1p (Fig. 1B,C). Region III is adjacent to the membranespanning domain, and region IV maps to the C-terminal end of
Nvj1p (Fig. 1B). These conserved regions helped guide our
efforts in mapping the putative functional domains of Nvj1p.
Region IV is the Vac8p-binding domain of Nvj1p
Protein-protein interactions between Nvj1p and Vac8p are
required for the formation of NV junctions. These interactions
are remarkable because they represent the only currently
known inter-organelle junction apparatus in eukaryotes
(Levine, 2004). Previously, we showed that Vac8p co-purifies
with 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). To
determine whether region IV is necessary for an association
with Vac8p, we deleted a cluster of conserved residues at the
C-terminal end of Nvj1p (aa 292-321; Fig. 1A,B) and analyzed
the localization of the truncated reporter (Nvj1p(293-321)EGFP) in the presence and absence of Vac8p. Normally in
VAC8+ cells, full-length Nvj1p-EGFP localizes to patches on
the nuclear surface that co-localize with FM4-64-stained
vacuole membranes, producing a yellow fluorescence
characteristic of NV junctions (Fig. 2A) (Pan et al., 2000). In
the absence of Vac8p, Nvj1p-EGFP diffuses evenly over the
surface 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 evenly
distributed 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

3624

Journal of Cell Science 119 (17)

Journal of Cell Science

B
I

II

III

IV

Nvj1p
aa
100-75%
74-50%

C
100

200

300

hydrophobic

2
1
0
-1
-2
-3
-4

hydrophilic
100

200

amino acid

immediate C-terminus of Nvj1p are necessary for binding

Vac8p and creating NV junctions. This Vac8p-binding region
is not required for the strict localization of Nvj1p to the nuclear
envelope, a property that must map elsewhere in the protein.
Finally, these results are consistent with our previous
observation that the C-terminal 40 residues of Nvj1p fused to
GFP partially localize to the vacuole membrane in a Vac8pdependent fashion (Pan et al., 2000).
Region I is necessary for anchoring Nvj1p to the ONM
The N-terminus of Nvj1p plays an important role in sorting
to the ONM, since the addition of tags onto its N-terminus
causes the mis-localization of Nvj1p to extranuclear ER-like

300

Fig. 1. Nvj1p can be divided into four domains

based on sequence homology across the
Saccharomyces family. (A) Alignment of S.
cerevisiae Nvj1p and its orthologs in closely
related and divergent Saccharomyces species.
Amino acid sequences were obtained from the
Saccharomyces Genome Database
(www.yeastgenome.org). The S. kluyveri
ortholog of Nvj1p was identified through a
tblastn search of contig 02.1344 (accession
AACE02000001). Sequences were aligned
using the ClustalX program (Thompson et al.,
1997). Boxes were drawn around regions of
sequence similarity in the Saccharomyces
Nvj1p family. (B) Graphical view of residues
in Nvj1p bearing similarity or identity across
the Saccharomyces Nvj1p family. ClustalX
conservation scores were calculated for each
column of the alignment in 1A, and residues
bearing similarity (50-74% homology, open
circles) or near identity (75-100%, shaded
diamonds) were aligned onto a linear map of
Nvj1p. These residues sorted independently
into four regions, as indicated. (C) KyteDoolittle hydropathy plot of the four conserved
regions of Nvj1p. Region I at the N-terminus
shows an area of considerable hydrophobicity,
while region II coincides with the membranespanning domain of Nvj1p.

membranes that form ectopic junctions with vacuoles (Kvam

and Goldfarb, 2004). The first ~30 residues of the N-terminus
(region I) comprise an imperfect hydrophobic sequence
bordered by basic residues, reminiscent of a signal sequence
(Fig. 1A,C) (Nielsen et al., 1997). Unlike signal peptide
sequences, which are post-translationally cleaved, neural
network algorithms predict that region I of Nvj1p is most likely
not cleaved (Bendtsen et al., 2004).
To investigate whether region I is a sorting determinant,
we constructed several truncations around its hydrophobic
sequence. Unlike full-length Nvj1p, which localizes strictly
to the ONM in both VAC8+ and vac8- cells (Fig. 2A), a
truncated reporter lacking the hydrophobic sequence of

Journal of Cell Science

Functional domains of Nvj1p

3625

Fig. 2. Tripartite signals mediate the targeting of Nvj1p

to NV junctions. (A) Region IV comprises the Vac8pbinding domain of Nvj1p. Full-length (FL) Nvj1p-EGFP
and a C-terminal truncation (293-321aa) lacking the
most conserved residues of region IV were localized in
vac8- and VAC8+ (nvj1-) cells. Nuclear chromatin
(blue) and vacuole membranes (red) were co-stained
with Hoechst and FM4-64, respectively. Interactions
between Nvj1p-EGFP and Vac8p on FM4-64-stained
vacuoles create NV junctions, which appear yellow in
the overlay. The C-terminal truncation of Nvj1p does
not form NV junctions and instead localizes uniformly
across the nuclear envelope. (B) Region I plays a role in
anchoring Nvj1p to the perinuclear ER. N-terminal
truncations preceding (1-6aa) or succeeding (1-26aa)
the hydrophobic sequence of region I, or eliminating the
N-terminus of Nvj1p entirely (1-86aa), were localized
in 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 ER
and form aberrant vacuole-associated membrane
junctions in the cytosol (see arrows) that appear
detached from the nucleus. (C) Region I and the
membrane-spanning region of Nvj1p (region II)
cooperatively sort to the perinuclear ER. Nvj1p
fragments tagged on their C-terminus with EGFP were
localized similarly to above. The membrane-spanning
region of Nvj1p (residues 87-120, denoted by a shaded
box) targets ER membranes but localizes exclusively to
the perinuclear ER in conjunction with region I (denoted
by a horizontal bar). Replacing the transmembrane
region of Nvj1p (between residues 90-121) with the first
transmembrane segment of Ste2p causes Nvj1p to
spread into the cortical ER and form aberrant junctions
with vacuoles in the cytosol (see arrows). Numbers in
diagram correspond to amino acid position.

region I (Nvj1p(1-26)-EGFP) spread into the cortical ER of

vac8- cells (Fig. 2B). Similar results were observed using a
reporter that lacked the entire N-terminus of Nvj1p
(Nvj1p(1-86)-EGFP) (Fig. 2B). These data demonstrate that
the N-terminus is not required for ER targeting but rather
functions after membrane insertion. When expressed in
VAC8+ cells, both truncated reporters formed atypical
junctions between or along the surface of vacuolar lobes that
appeared spatially detached from the nucleus in most cells
(Fig. 2B). Thus, when bound to Vac8p, these N-terminally
truncated reporters no longer escaped to the cortical ER but
rather formed extranuclear ER junctions with vacuoles.
Conversely, deleting the first six residues of Nvj1p did not
significantly affect the targeting of Nvj1p(1-6)-EGFP to the
ONM in vac8- cells, although a low level of cortical staining
was sometimes visible (Fig. 2B). Moreover, Nvj1p(1-6)EGFP formed normal perinuclear NV junctions in VAC8+ cells
rather than aberrant extranuclear junctions (Fig. 2B). Thus,
residues upstream of the hydrophobic sequence in region I are
not critical for ONM sorting. In total, these experiments
directly implicate the hydrophobic signal anchor-like
sequence of Nvj1p in proper NV-junction formation by

targeting the ONM. Interestingly, a reporter containing the

first 90 aa of Nvj1p fused to GFP localized to mitochondria
(our unpublished results), which is consistent with the fact that
region I of Nvj1p resembles the signal-anchor sequences of
the outer mitochondrial membrane proteins Tom20p and
Tim70p (Waizenegger et al., 2003).
The membrane-spanning domain of Nvj1p is also
required for ONM targeting
As described above, deleting the first 86 residues of Nvj1p did
not prevent targeting of this reporter to the ER (Fig. 2B). Since
this deletion also retains C-terminal associations with the
cytoplasmic vacuoles in VAC8+ cells, the N-terminus of Nvj1p
is not required for properly orienting the protein in the
membrane. Further truncation experiments revealed that a
fragment of Nvj1p (aa 87-120) corresponding to the
membrane-spanning domain of Nvj1p (region II; Fig. 1B,C)
was sufficient to localize throughout the perinuclear and
cortical ER network (Fig. 2C). Thus, region II of Nvj1p
contains sufficient sequence information for targeting ER
membranes rather than the plasma membrane or other
organelles. Importantly, an N-terminal fragment of Nvj1p

3626

Journal of Cell Science 119 (17)

Journal of Cell Science

comprising both regions I and II (Nvj1p(121-321)-EGFP)

localized strictly to the nuclear envelope (Fig. 2C). These data
illustrate that the hydrophobic N-terminal signal anchor-like
sequence and the membrane-spanning domain of Nvj1p work
in concert to mediate targeting to the ONM. To test whether
the membrane-spanning domain of another protein can
function in this regard, we replaced residues 90-120 of Nvj1p
with a slightly modified version of the first membranespanning domain of Ste2p, which has been used extensively
to study co-translational insertion into the ER (see Harley and
Tipper, 1996). As shown in Fig. 2C, Nvj1p(Ste2pTM)-EGFP
aberrantly spread into the cortical ER of vac8- cells despite
the presence of a native N-terminal domain (region I).
Likewise, this chimeric reporter formed aberrant, vacuoleassociated membrane junctions in VAC8+ cells (Fig. 2C),
similar to those observed in cells expressing N-terminal
truncations of Nvj1p (Fig. 2B). In summary, these data
indicate that regions I and II function co-operatively to target
Nvj1p into the ONM, and that the membrane-spanning
domain of Nvj1p is adapted for this purpose.
Blocking the N-terminus of Nvj1p leads to ONMexpansion and separation from the INM
As described above, region I is required for the strict
localization of Nvj1p to the ONM. It is possible that this
domain functions to anchor Nvj1p in the ONM by attaching to
the INM. By analogy, the ONM localization of KASH-domaincontaining nesprin isoforms is mediated by interactions across
the perinuclear lumen with SUN domain proteins (Crisp et al.,
2006). A bridging model for Nvj1p is consistent with the
observation that deleting its N-terminus results in the formation
of extranuclear junctions in VAC8+ cells (Fig. 2B). Likewise,
we previously reported that blocking the N-terminus of Nvj1p
with a 3HA epitope promoted the formation of analogous
extranuclear junctions (Kvam and Goldfarb, 2004). If our
bridging model is true, then extranuclear ER-vacuole junctions
might arise from sections of the ONM that have become
detached from the INM.
To test this hypothesis we employed an N-terminally fused
GFP-Nvj1p reporter that, like N-terminal truncations (Fig. 2B),
spread into the cortical ER of vac8- cells and promoted the
formation of extranuclear ER junctions with vacuoles in
VAC8+ cells (Fig. 3A). Anti-GFP immuno-EM was used to
characterize the GFP-Nvj1p-labeled membranes associated
with these extranuclear junctions. The example shown in Fig.
3B shows an extension of the ONM that is sandwiched between
two vacuole lobes. The presence of GFP-Nvj1p in these
membranes is confirmed by the presence of anti-GFP colloidal
gold labeling (Fig. 3B, asterisks). A striking separation
between the outer- and inner-nuclear membranes is apparent
by the triangular expansion of the perinuclear lumen, which,
as expected, is devoid of electron dense material such as
ribosomes. These results are consistent with the hypothesis that
the N-terminus of Nvj1p mediates ONM-localization through
a direct or indirect physical attachment with the INM. A yeast
two-hybrid screen for INM proteins that might interact with the
N-terminal domain of Nvj1p failed to identify potential
candidates (our unpublished results). It is also possible that the
N-terminal domain of Nvj1p inserts directly into the lipid
bilayer of the INM, by analogy to the insertion of the Nterminal amphipathic helix of Sar1p into the ER (Lee et al.,

2005). Our results are also interesting because they show that
zipper-like interactions between Nvj1p and Vac8p can promote
membrane expansion in a nuclear envelope subdomain (see
Campbell et al., 2006).
Region II of Nvj1p sequesters the ER membrane protein
Tsc13p
We previously demonstrated Tsc13p, an ER membrane protein
involved in VLCFA biosynthesis, is recruited into NV
junctions through associations with Nvj1p (Kvam et al., 2005).
Using confocal microscopy, we mapped the region of Nvj1p
responsible for interacting with Tsc13p. Briefly, N- and Cterminal truncations of Nvj1p were tested for their ability to
sequester Tsc13p-EYFP from the cortical ER to the nuclear
envelope 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 in
vac8- cells, and accumulates in extranuclear junctions between ERlike 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) Immunogold
localization of GFP-tagged Nvj1p by electron microscopy. GFPtagged Nvj1p reporters were induced prior to cryofixation and
immuno-EM analysis as described in Materials and Methods. Nvj1pGFP, a functional reporter, localizes to NV junctions. Colloidal-goldconjugated antibodies against GFP (asterisk) label the surface of
nuclei at NV junctions. By contrast, GFP-Nvj1p (a non-functional
reporter) forms extranuclear junctions with vacuoles. Here, colloidalgold-conjugated antibodies against GFP (asterisk) label an extension
of the ONM (black arrows) that is sandwiched between two vacuole
lobes. The ONM is separated from the INM (denoted by white
arrowheads), forming an extensive void in the perinuclear lumen. N,
nuclei; V, vacuoles. Bars, 0.3 m.

Functional domains of Nvj1p

separate Tsc13p-binding activity. However, we determined that

region II alone is sufficient to co-immunoprecipitate Tsc13pEYFP in vivo. As shown in Fig. 4B, Tsc13p-EYFP was
efficiently co-immunoprecipitated from detergent-extracted
cell lysates using a 3HA-tagged fragment of region II
containing the membrane-spanning domain of Nvj1p and some
bordering residues (Nvj1p(87-120)-3HA). Together, these
results indicate that the sequences within region II are
sufficient for binding and sequestering Tsc13p in the ER. By
analogy, this interaction may occur between the membranespanning domains of these two proteins or possibly among
residues very close to the plane of the membrane.

Journal of Cell Science

membrane-spanning domain (region II) of Nvj1p proved

dispensable for an association with Tsc13p (Fig. 4A). Tsc13p
was efficiently sequestered to the nuclear envelope upon
expression 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 (aa
87-321) lacking residues preceding region II was sufficient to
sequester Tsc13p into extranuclear, vacuole-associated
membrane junctions (Fig. 4A), which arose as a consequence
of the mis-targeting of this reporter (Fig. 2B). These data
collectively narrowed down the Tsc13p-interaction domain to
the membrane-spanning domain (region II) of Nvj1p.
Alternatively, it was possible that both fragments contained

3627

Fig. 4. Region II of Nvj1p sequesters Tsc13p. (A) The membranespanning domain of Nvj1p (region II) is responsible for recruiting
Tsc13p-EYFP. Full-length (FL) or N- and C-terminal truncations of
Nvj1p were overexpressed in nvj1- cells and tested for the ability to
sequester Tsc13p-EYFP from cortical ER compartments to the
perinuclear 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 the
sequestration of Tsc13p-EYFP to the nuclear envelope. Numbers
correspond to amino acid positions. (B) The transmembrane region
of Nvj1p (aa 85-120) is sufficient to co-immunoprecipitate Tsc13pEYFP in vivo. Detergent-extracted lysates of cells expressing
Tsc13p-EYFP and 3HA-tagged Nvj1p(85-120aa) or empty vector
were prepared as described in Materials and Methods. Nvj1p(85120aa)-3HA was immunoprecipitated (IP) with anti-HA conjugated
agarose beads.

Fig. 5. Region III of Nvj1p is necessary and sufficient to bind Osh1p.

(A) Deletion of the most conserved residues in region III abrogates
the Nvj1p-dependent sequestration of GFP-Osh1p to the nuclear
envelope. Full-length (FL) or N- and C-terminal truncations of Nvj1p
were overexpressed in nvj1- cells and tested for the ability to
sequester GFP-Osh1p from soluble and Golgi-associated pools to the
nuclear surface. Conserved residues in region III of Nvj1p (aa 130176) proved necessary for GFP-Osh1p sequestration. Numbers
correspond to amino acid positions. (B) Residues 130-177 of Nvj1p
are sufficient to co-immunoprecipitate GFP-Osh1p in vivo. Lysates
of cells expressing GFP-Osh1p and 3HA-tagged Nvj1p(130-177aa)
or empty vector were prepared as described in Materials and
Methods. Nvj1p(130-177aa)-3HA was immunoprecipitated (IP) with
anti-HA conjugated agarose beads.

Journal of Cell Science

3628

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Region III is sufficient and necessary to bind Osh1p

We previously showed that Nvj1p sequesters Osh1p into NV
junctions in a Vac8p-independent fashion, most likely by direct
binding (Kvam and Goldfarb, 2004). In order to identify the
region of Nvj1p responsible for interacting with Osh1p, we
expressed 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 by
confocal microscopy. As shown in Fig. 5A, the putative Osh1pbinding site mapped to a segment of Nvj1p (aa 120-177)
adjacent to the membrane-spanning domain. Importantly, this
area coincided with region III of Nvj1p (Fig. 1A,B) and, like
the Vac8p-binding domain (region IV), is exposed to the
cytoplasm. Saccharomyces orthologs of Nvj1p contain an
especially well conserved sequence between residues 140-174
in region III (Fig. 1A,B). We created a myc-tagged Nvj1p
mutant with an internal deletion that eliminated these
conserved residues. Expression of this mutant, Nvj1p(130176)-myc, was confirmed by immunoblot (our unpublished
data). As expected, overexpression of Nvj1p(130-176)-myc
failed to sequester GFP-Osh1p from surrounding cytoplasmic
and Golgi compartments (Fig. 5A). This fact was not due to
improper targeting of the Nvj1p(130-176)-myc reporter, since
a 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 cortical
ER to NV junctions (our unpublished data). In biochemical
pull-down experiments, GFP-Osh1p efficiently coimmunoprecipitated with an epitope-tagged fragment of region
III encompassing residues 130-177 of Nvj1p (Nvj1p(130177)-3HA) (Fig. 5B). Together, these results show that a
conserved sequence within region III is both necessary and
sufficient for an interaction between Nvj1p and Osh1p.

NVJ1-overexpressing cells exhibit a cold-sensitive

tryptophan transport defect related to Osh1p
Several studies have demonstrated that deleting OSH1 in
tryptophan auxotrophs (trp1) produces a temperature-sensitive
cell 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 is
likely related to the sterol-dependent sorting of the highaffinity 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 into
detergent-resistant membrane domains (DRMs, or lipid rafts)
within late Golgi or post-Golgi compartments (Umebayashi
and Nakano, 2003; Gaber et al., 1989). Based on these results,
we reasoned that the overexpression of Nvj1p might
cause an analogous slow-growth phenotype by
sequestering Osh1p into NV junctions and away from the
trans-Golgi. Indeed, compared to control cells expressing
an empty vector, NVJ1-overexpressing cells were
hypersensitive to tryptophan limitation at low
temperature (Fig. 6A). This growth defect was
complemented by excess tryptophan or higher
temperature (Fig. 6A), analogous to what was observed
in osh1- cells (Levine and Munro, 2001). Moreover, the
growth defect of NVJ1-expressing cells was rescued by
single copy CEN plasmids expressing either TAT1 or
TAT2 tryptophan permeases under the control of their
Fig. 6. Overexpression of Nvj1p, but not a mutant form
lacking Osh1p-binding activity, confers a tryptophan
transport-related growth defect. (A) Cells overexpressing
Nvj1p, but not a mutant form lacking the Osh1p-binding
domain, grow poorly on media containing limiting
concentrations of tryptophan at low temperature. Cells
harboring PCUP1-NVJ1, PCUP1-NVJ1(130-176aa)-myc, or
empty vector were induced and replica-plated onto media
containing limiting (15 g/ml) or excess (40 g/ml)
tryptophan as described in Materials and Methods. (B) The
cold-sensitive growth phenotype of Nvj1p-overexpressing
cells is suppressed by low- and high-affinity tryptophan
permeases. Cells harboring PCUP1-NVJ1 and either pTAT1,
pTAT2, or empty vector were induced and replica-plated onto
media containing limiting (15 g/ml) or excess (40 g/ml)
tryptophan as above. (C) NV junctions are not necessary to
confer tryptophan transport defects in Nvj1p-overexpressing
cells. Growth curves of vac8- cells expressing PCUP1-NVJ1
(open squares), PCUP1-NVJ1(130-176aa)-myc (black
triangles), or empty vector (gray circles) were determined in
liquid media containing limiting (15 g/ml) or excess (40
g/ml) tryptophan at 25C using a Bioscreen C Analyzer (see
Materials and Methods).

Functional domains of Nvj1p

NVJ1+ trp1 cells grow poorer in tryptophan-depleted

media than nvj1- trp1 cells
Osh1p localizes to both trans-Golgi membranes and NV
junctions, but is found almost exclusively at NV junctions as
Nvj1p levels rise by ectopic overexpression or as a
consequence of nutrient depletion during late-log phase (Kvam
and Goldfarb, 2004; Levine and Munro, 2001). Because high
levels of Nvj1p affect cell growth by sequestering Osh1p (Fig.
6), we were interested in determining whether physiologically
relevant levels of Nvj1p were active in this regard. For this
purpose, we analyzed the growth of isogenic NVJ1+ trp1 and
nvj1- trp1 cells as a function of tryptophan limitation. At

5 g/ml Trp

1.2

OD600

1
0.8
0.6
0.4
0.2

wt

nvj1

0
0

10

15

20

25

30

35

Time (hrs)

15 g/ml Trp

1.4
1.2
1

OD600

Journal of Cell Science

native promoters (Fig. 6B). By contrast, cells overexpressing

Nvj1p(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 cell
growth rates at 25C in liquid media containing low or high
concentrations of tryptophan (Fig. 6C). The ability of Nvj1p to
form NV junctions proved to be unimportant for these
tryptophan transport-related growth phenotypes, since similar
results were observed in vac8- cells (Fig. 6C). In summary,
these results demonstrate that the sequestration of Osh1p by
Nvj1p causes a defect in tryptophan uptake similar to that
previously reported for osh1- cells.

0.8
0.6
0.4
0.2

wt

nvj1

0
0

10

15

20

25

30

35

Time (hrs)

Fig. 7. Native levels of Nvj1p are mildly deleterious for growth in

tryptophan-depleted media. nvj1- and its isogenic parental (NVJ1+)
tryptophan auxotrophic strain (trp1) were grown in rich media to logphase and shifted into SC media containing limiting (15 g/ml) or
depleted (5 g/ml) concentrations of tryptophan. Cell growth was
monitored at 25C using a Bioscreen C Analyzer (see Materials and
Methods). Error bars were calculated from two independent trials
performed in duplicate.

3629

concentrations of tryptophan sufficient to slow the growth of

NVJ1-overexpressing cells (15 g/ml), the growth of NVJ1+
and nvj1- cells at 25C was indistinguishable (Fig. 7B).
However, nvj1- cells grew markedly better than NVJ1+ cells
when the concentration of extracellular tryptophan became
increasingly 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 may
be mildly deleterious for growth in tryptophan-depleted
environments by affecting the localization and activity of
Osh1p.
Discussion
Nvj1p is a remarkable protein that mediates several novel
phenomena, including the formation of the only known interorganelle membrane junction apparatus (NV junctions), and a
unique type of autophagy that pinches-off and degrades nonessential parts of the yeast nucleus (PMN). The key findings
of this study include mapping the regions of Nvj1p responsible
for sorting to the ONM and interacting with its bindingpartners, 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 to
four regions (I-IV) of Nvj1p that are conserved among the
Saccharomyces family of NVJ1 orthologs. Some of these
sequence regions are present in increasingly divergent Nvj1plike proteins in more evolutionarily distant yeasts, including
Kluveromyces waltii, Kluveromyces lactis, Candida glabrata
and Ashbya gossypii (not shown). It should be noted that the
genomes of all fungal species whose genomes have been
sequenced, including Aspergillis nidulans and Neurospora
crassa, encode well-conserved orthologs of VAC8, TSC13 and
OSH1. Our results also reveal a novel role for Nvj1p in the
regulation of tryptophan transport. These experiments are the
first to expose a growth defect associated with NVJ1
expression. From our results, we propose that the sequestration
of Osh1p by Nvj1p inhibits tryptophan uptake from the
environment, possibly by modulating tryptophan permeasetrafficking to the plasma membrane via a mechanism that was
previously shown to require Osh1p (Levine and Munro, 2001;
Jiang et al., 1994).
The sorting of eukaryotic proteins to the ONM is usually
explained in terms of binding to resident proteins of the nuclear
envelope, which begs the question of how the resident proteins
themselves become localized. We show that the N-terminal
hydrophobic domain (region I) and the membrane-spanning
domain (region II) of Nvj1p are both required for efficient
localization to the ONM. Region I contains a signal anchorlike sequence consisting of a relatively short, imperfect
hydrophobic 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 Nterminal truncations of Nvj1p retain C-terminal associations
with both Osh1p and Vac8p in the cytoplasm. The cytoplasmic
C-terminal domain of Nvj1p (aa 121-321) is dispensable for
ONM sorting, which rules out a role for Vac8p or Osh1p in
this regard. However, Nvj1p-truncations lacking region I fail
to localize strictly to the ONM and, instead, are found
ubiquitously throughout the perinuclear and cortical ER. The
membrane-spanning domain within region II plays an
additional role in sorting to the ONM and, as discussed below,

Journal of Cell Science

3630

Journal of Cell Science 119 (17)

also interacts with Tsc13p. A 47-residue segment of region II

is sufficient to target a GFP reporter to the ER network. We
found that the important function of this domain in ONM
sorting cannot be substituted by the first membrane-spanning
domain of Ste2p, despite the fact that this chimera was
efficiently 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 on
Vac8p (Pan et al., 2000). We mapped the Vac8p-binding
domain of Nvj1p to its C-terminus (region IV), which is
consistent with previous two-hybrid results (Pan et al., 2000).
When the sorting of Nvj1p is altered (either by blocking or
mutating region I or through substitution of region II), Nvj1p
spreads into the cortical ER and forms aberrant junctions with
Vac8p-associated vacuole membranes. As shown in Fig. 3,
these extranuclear junctions originate as extensions of the
ONM. 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 ONM
and prevent its escape into intermediate and cortical ER
compartments. A physical connection to the INM would also
explain how the ONM and INM move in concert into vacuole
invaginations during PMN (Roberts et al., 2003). In support of
this notion, blocking the N-terminus of Nvj1p with GFP
promotes the separation of the inner and outer nuclear
membranes, leading to expansion of the ONM through
multiple zipper-like interactions between Nvj1p and Vac8p on
the vacuole surface. Precedent for a nuclear envelope bridging
apparatus comes from recent reports describing the LINC
complex, which involves interactions between KASH-domaincontaining nesprin isoforms in the ONM with SUN domain
proteins 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 protein
in the INM, since the depletion or the secretion of SUN
domains 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 the
membrane-spanning domain of Nvj1p interacts with one or
more of the membrane-spanning domains of Tsc13p within the
ER bilayer. Associations among membrane-spanning domains

vacuole membrane

ii

Vac8p

IV

Osh1p III

ONM

Osh1p III

Tsc13p

II

I
INM

Tsc13p

II

ONM

X
INM

vacuole membrane

Vac8p

IV

are known to stabilize certain integral membrane protein

complexes (Li et al., 2003). Interactions between Nvj1p and
Tsc13p, which are likely to be direct, may occur within the
hydrophobic interior of the membrane or via residues
immediately flanking their membrane-spanning segments. In
this regard, it is interesting that the co-localization of Tsc13p
with 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 by
Levine and Munro (Levine and Munro, 2001), is mediated by
Vac8p-independent interactions with Nvj1p (Kvam and
Goldfarb, 2004). Osh1p is particularly interesting because of
its likely role in trafficking the high-affinity tryptophan
permease, Tat2p. Tat2p is a constitutively expressed membrane
permease that is routed to either the plasma membrane or
vacuole depending on the concentration of tryptophan in the
medium (Beck et al., 1999). A recent study revealed that Tat2p
trafficking is dependent on its association with detergentresistant membrane domains (DRMs) in post-Golgi
compartments (Umebayashi and Nakano, 2003). In fact,
several mutations disrupting the synthesis of DRM-associated
lipids 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 et
al., 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]tryptophan
uptake and osh1- trp1 cells exhibit a severe growth defect on
low tryptophan media (Levine and Munro, 2001; Jiang et al.,
1994). Taken together, these published results strongly
implicate Osh1p as a key player in the sterol-dependent
trafficking of Tat2p to the plasma membrane.
Here, we report that overexpression of Nvj1p in trp1
auxotrophs causes a slow-growth phenotype similar to that
reported for osh1- cells (Levine and Munro, 2001). This
growth defect is dependent on the sequestration of Osh1p,
since cells overexpressing a mutant version of Nvj1p without
an Osh1p-binding domain (region III) grew normally. The
Nvj1p-dependent sequestration of Osh1p may provide a ready
mechanism to downregulate tryptophan permease activity in
response to nutrient depletion. In support of this notion, native
levels of Nvj1p confer a moderating effect on cell growth in
low-tryptophan media, presumably due to the steady (but
physiologically significant) sequestration of Osh1p. As cells

Fig. 8. Molecular model of NV junctions. Nvj1p is restricted to

the outer nuclear membrane, which is continuous with the
perinuclear ER. The membrane-spanning domain (II) of Nvj1p
interacts with Tsc13p in the ER membrane. An adjacent
cytosolic region (III) associates with Osh1p. The C-terminal
tail of Nvj1p (IV) binds to Vac8p, which is acylated to the
vacuole membrane. The hydrophobic N-terminus of Nvj1p (I)
may link the inner- and outer-nuclear membranes either by
binding an unknown factor (X) in the INM (panel i), or by
inserting directly into the INM by spanning across the
perinuclear lumen (panel ii).

Journal of Cell Science

Functional domains of Nvj1p

progress into late log phase, GFP-Osh1p becomes increasingly
associated with NV junctions and less so at trans-Golgi
compartments (Levine and Munro, 2001) (our unpublished
results). It is during late log phase when high-affinity nutrient
permeases such as Tat2p are routed to the vacuole and degraded
(Schmidt et al., 1998). These observations are consistent with
the hypothesis that Nvj1p is a negative regulator of Osh1p
function and, as such, suggest that Nvj1p plays a novel role in
sterol-dependent protein trafficking. Previous studies have also
implicated the yeast homolog of mammalian VAMP-associated
protein, Scs2p, in targeting Osh1p and other yeast ORPs to
cellular membranes, although Scs2p is not strictly required for
the 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 control
Osh1p function.
The faster growth of nvj1- trp1 cells in limiting tryptophan
suggests that Nvj1p may normally function as a finely tuned
rheostat to control Osh1p activity, even during log phase when
Nvj1p levels are normally low. Our results may also account
for the increased survival of nvj1- cells during stationary
phase, which is a function of chronological aging (see Fabrizio
et al., 2005). A recent genome-wide screen identified mutants
in NVJ1 and a number of genes implicated in nutrient sensing
and TOR signaling as some of the longest-lived in the deletion
collection (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 and
Thompson, 2002; Beck et al., 1999; Schmidt et al., 1998).
In conclusion, we have mapped several functional domains
of Nvj1p that mediate the recruitment of Vac8p, Osh1p and
Tsc13p to the ONM, and revealed roles for both the N-terminal
signal anchor-like sequence and membrane-spanning domain
of Nvj1p in sorting to the ONM. Finally, we describe a
functional link between the sequestration of Osh1p into NV
junctions and the regulation of sterol-dependent protein
trafficking to the plasma membrane. This mechanism may have
broader implications for the control of ORP function in higher
cells.
Materials and Methods
Yeast strains and growth conditions
Yeast strains used in this study were based in the YEF473 genetic background (trp163 leu2-1 ura3-52 his3-200 lys2-801) (Bi and Pringle, 1996). Deletion of NVJ1
and VAC8 in YEF473 was described elsewhere (Pan et al., 2000; Pan and Goldfarb,
1998). Unless otherwise indicated, cells were cultured at 30C in YPD, synthetic
complete media (SC), or standard dropout media containing 2% glucose (Sherman,
1991).

Plasmids
Plasmids for the expression of NVJ1, NVJ1-GFP or GFP-NVJ1 under CUP1
promoter control (PCUP1-NVJ1, PCUP1-NVJ1-GFP, PCUP1-GFP-NVJ1) were
described previously (Pan et al., 2000). Nvj1p truncations were constructed by PCRamplifying fragments of NVJ1 corresponding to the indicated amino acid positions
with primers that introduced EcoRI and HindIII sites. These fragments were ligated
into the EcoRI and HindIII sites of pEGFP-C-FUS, which was constructed from
pGFP-C-FUS (Niedenthal et al., 1996) by replacing GFP with a PCR-amplified
ClaI-EGFP-SalI fragment generated from pEGFP (Clontech, CA). To express nonfluorescent versions of these truncations, NVJ1 fragments were subcloned from
pEGFP-C-FUS into the PGAL1/10 plasmid pESC(HIS) (Stratagene) using EcoRI and
ClaI sites. For immunoprecipitation experiments, the FLAG epitope of pESC(HIS)
was replaced with the triple HA epitope from pGTEPI (Tyers et al., 1992) using
SpeI and SacI sites, and PCR-amplified fragments of NVJ1 were cloned upstream
of 3HA using EcoRI and SpeI sites. Deletion of residues 130-176 of Nvj1p was
accomplished by fusing two PCR-amplified fragments of NVJ1, corresponding to

3631

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). To
replace the membrane-spanning region of Nvj1p with that of Ste2p, an XholIsubstituted chimeric NVJ1/STE2 reverse primer (5-ccgctcgaggacaatcaaagtcaaagcagctgcaccacatatgacaccaaacataatggccaattcgctggattgtctgg-3), corresponding to
residues 84-90 of Nvj1p and residues 52-68 of Ste2p [including the mutation R58I
used by Harley and Tipper (Harley and Tipper, 1996)], was used to PCR-amplify a
fragment corresponding to the first 90 residues of NVJ1. This chimeric fragment
was ligated to a second NVJ1 PCR fragment (corresponding to residues 121-321)
using introduced XholI sites, and cloned into pEGFP-C-fus. Plasmids expressing
chimeric or truncated version of NVJ1 were confirmed by sequencing. Plasmids for
expression of the tryptophan permease genes TAT1 and TAT2 (pTAT1 and pTAT2)
were generously supplied by M. Hall (Schmidt et al., 1994). Plasmids for the
constitutive expression of GFP-Osh1p (pRS416-GFP-OSH1) or the inducible
expression 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- cells

Growth sensitivity on solid media was assayed using previously described methods
for tryptophan uptake (Levine and Munro, 2001). Briefly, log-phase trp1 cells
harboring the indicated PCUP1 plasmids were induced for 3 hours with 0.1 mM
CuSO4. Three OD600 units of culture were then collected by centrifugation. Cell
pellets were resuspended in 1 ml of fresh media containing 0.1 mM CuSO4, diluted
serially by tenfold, and plated onto selective media containing limiting (15 g/ml)
or excess (40 g/ml) concentrations of tryptophan using a multi-pronged replicator.
Plates were incubated at 25C or 37C for 72-96 hours. For growth-curve analyses
in liquid media, log-phase trp1 cells harboring the indicated PCUP1 plasmids were
induced for 3 hours with 0.1 mM CuSO4. A total of 0.25 OD600 units of culture
were collected by centrifugation and inoculated into 5 ml of SC media containing
limiting (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 a
Honeycomb 2 cuvette multiwell plate (MTX Lab Systems, Vienna, VA). Cells were
grown at 25C in a Bioscreen C Analyzer (MTX Lab Systems) programmed to
measure 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 were
collected using EZExperiment software (MTX Lab Systems). Triplicate readings
were averaged for each strain. A similar protocol was used to measure the growth
of nvj1- and its parental trp1 strain in SC media containing 5 g/ml or 15 g/ml
of tryptophan.

Immunoprecipitation and immunoblotting

Cells 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-TSC13EYFP were cultured in selective media containing 2% raffinose to log-phase.
Protein expression was induced with 2% galactose or, where appropriate, 0.1 mM
CuSO4 for 3.5 hours. Approximately 50 OD600 units of culture were collected by
centrifugation. Cell pellets were weighed and overlayed with an equivalent volume
of acid-washed 425-600 m glass beads (Sigma) and 2 volumes of extraction
buffer (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 2
g/ml pepstatin A, aprotinin, and leupeptin (Sigma). Cells were vortexed five times
for 2 minutes, intermittent with 2 minute incubations on ice. Lysate suspensions
were solubilized with 1% NP-40, vortexed, and cleared by centrifugation (500 g
for 10 minutes). After collecting the supernatant, glass beads were washed with 2
volumes of EB, vortexed, and cleared by centrifugation. Combined supernatants
were incubated with 30 l of goat IgG-agarose for 15 minutes with rotation to
remove non-specific peptides. After brief centrifugation, the total protein
concentration 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 when
necessary. Lysates were incubated with 30 l of goat anti-HA conjugated agarose
at 4C overnight with rotation (Santa Cruz Biotechnology). Bead complexes were
isolated by centrifugation (500 g for 1 minute), washed four times with HNTG (20
mM HEPES pH 7.5, 0.15 M NaCl, 0.1% Triton X-100, 10% glycerol), and eluted
into a fresh tube with 35 l of 2 protein gel sample loading buffer (100 mM TrisHCl pH 6.8, 2% SDS, 20% glycerol, 2% 2-mercaptoethanol, 0.1% bromophenol
blue). Eluates were boiled for 5 minutes, and 10-15 l of sample were analyzed
by 8% and 18% SDS-PAGE. GFP-Osh1p and Tsc13p-EYFP were probed by
Immuno blot with polyclonal BD Living ColorsTM A.v. peptide antibodies
(Clontech). HA-tagged Nvj1p fragments were probed with mouse anti-HA
monoclonal antibodies (Santa Cruz Biotechnology). All immuno-probed proteins
were detected using alkaline phosphatase-coupled goat anti-rabbit IgG (Santa Cruz
Biotechnology) or alkaline phosphatase-coupled goat anti-mouse IgG (Zymed) and
developed colometrically

Cell imaging and confocal microscopy

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

3632

Journal of Cell Science 119 (17)

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, Tsc13pEYFP 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 was
performed 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 microscopy

Journal of Cell Science

Wild-type cells harboring PCUP1-NVJ1-GFP or PCUP1-GFP-NVJ1 were grown to log

phase 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 electron

microscopy, and Xiaozhou Ryan for the confocal images in Fig. 3.
We also thank Rebecca Gilson for assistance with tryptophan uptake
assays, Michael Hall for providing pTAT1 and pTAT2 plasmids, Tim
Levine for providing pRS416-GFP-OSH, and Bill Burke for helpful
comments on the manuscript. This study was supported by US
National Institutes of Health RO1 grant GM67838 (to D.S.G.).

References
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