Research Article
4495
Intramolecular protein-protein and protein-lipid
interactions control the conformation and subcellular
targeting of neuronal Ykt6
Haruki Hasegawa1, Zhifen Yang1, Leif Oltedal2, Svend Davanger3 and Jesse C. Hay1,*,‡
1University
2University
3University
of Michigan, Department of Molecular, Cellular and Developmental Biology, Ann Arbor, MI 48109-1048, USA
of Bergen, Department of Anatomy and Cell Biology, Locus on Neuroscience, 5009 Bergen, Norway
of Oslo, Centre for Molecular Biology and Neuroscience, PO Box 1105 Blindern, 0317 Oslo, Norway
*Present address: University of Montana, Division of Biological Sciences, COBRE Center for Structural and Functional Neuroscience, 32 Campus Drive, Missoula, MT 59812-4824,
USA
‡Author for correspondence (e-mail: [email protected])
Accepted 12 May 2004
Journal of Cell Science 117, 4495-4508 Published by The Company of Biologists 2004
doi:10.1242/jcs.01314
Summary
Although the membrane-trafficking functions of most
SNAREs are conserved from yeast to humans, some
mammalian SNAREs have evolved specialized functions
unique to multicellular life. The mammalian homolog of
the prenylated yeast SNARE Ykt6p might be one such
example, because rat Ykt6 is highly expressed only in
brain neurons. Furthermore, neuronal Ykt6 displayed a
remarkably specialized, punctate localization that did not
overlap appreciably with conventional compartments of
the endomembrane system, suggesting that Ykt6 might be
involved in a pathway unique to or specifically modified for
neuronal function. Targeting of Ykt6 to its unique
subcellular location was directed by its profilin-like longin
domain. We have taken advantage of high-resolution
structural data available for the yeast Ykt6p longin domain
to examine mechanisms by which the mammalian longin
domain controls Ykt6 conformation and subcellular
targeting. We found that the overall tertiary structure of
the longin domain, not sequence-specific surface features,
drives direct targeting to the Ykt6 punctate structures.
However, several sequence-specific surface features of
the longin domain indirectly regulate Ykt6 localization
Introduction
Soluble N-ethylmaleimide-sensitive-factor attachment protein
receptors (SNAREs) are central components of the
intracellular membrane fusion machinery (for reviews, see
Hay, 2001; Ungar and Hughson, 2003). Many mammalian
SNAREs have yeast homologs with similar functions but
others have evolved more specialized roles in specific tissues
with specific membrane trafficking needs. For example,
syntaxin 1A, SNAP-25 and VAMP2 are SNAREs involved in
synaptic-vesicle exocytosis that lack direct counterparts in
yeast. It is not yet clear, however, what the specialized
functions of certain tissue-specific SNAREs are. For example,
we do not know why muscle cells need VAMP5 on their
plasma membranes (Zeng et al., 1998), what the function of
syntaxin 17 is in the smooth endoplasmic reticulum (ER) of
steroidogenic cells (Steegmaier et al., 2000) or what the
apparently specialized role of syntaxin 11 is in the immune
through intramolecular interactions that mask otherwisedominant targeting signals on the SNARE motif and lipid
groups. Specifically, two hydrophobic binding pockets, one
on each face of the longin domain, and one mixed
hydrophobic/charged surface, participate in proteinprotein interactions with the SNARE motif and proteinlipid interactions with the lipid group(s) at the molecule’s
C-terminus. One of the hydrophobic pockets suppresses
protein-palmitoylation-dependent mislocalization of Ykt6
to the plasma membrane. The Ykt6 intramolecular
interactions would be predicted to create a compact, closed
conformation of the SNARE that prevents promiscuous
targeting interactions and premature insertion into
membranes. Interestingly, both protein-protein and
protein-lipid interactions are required for a tightly closed
conformation and normal targeting.
Supplemental data available online at http://jcs.biologists.org/cgi/
content/full/117/19/4495/DC1
Key words: SNAP receptor, SNARE, Ykt6, Vesicle transport, Protein
prenylation, Protein palmitoylation
system (Prekeris et al., 2000). In addition, some SNAREs [e.g.
syntaxin 16 (Dulubova et al., 2002)] are selectively highly
enriched in brain but are probably not involved in regulated
exocytosis.
Mammalian Ykt6 is another brain SNARE likely to have
unexpected function(s). Yeast Ykt6p appears to be involved in
multiple transport pathways between the Golgi and vacuole
(Dilcher et al., 2001; Tsui and Banfield, 2000), and is required
for homotypic vacuole fusion (Ungermann et al., 1999). Hence,
although yeast YKT6 is an essential gene, it does not appear to
have a single function in yeast. In fact, it appears to be an
example of a multifunctional member of the R-SNARE family
(Fasshaur et al., 1998), perhaps partially overlapping in
function with other R-SNAREs such as Sec22p and Nyv1p. In
support of this idea, Ykt6p expression was specifically
upregulated in Sec22p-lacking strains and appeared to
compensate partially for the Sec22p deletion by participating
4496
Journal of Cell Science 117 (19)
in ER-to-Golgi SNARE complexes normally containing
Sec22p (Liu and Barlowe, 2002).
Mammalian Ykt6 might have even more diverse roles,
because it has several novel and unexpected features. For
example, rat Ykt6 is highly enriched in brain (Hasegawa et al.,
2003). This finding was surprising, because a SNARE involved
in constitutive biosynthetic transport to the lysosome (as yeast
Ykt6p is) would probably be very widely expressed.
Furthermore, Ykt6 was localized to punctate vesicular
structures spread throughout the cytoplasm that did not
colocalize with established markers of the endomembrane
system. These findings implied that mammalian Ykt6 might
function in a specialized transport pathway in neurons instead
of or in addition to a constitutive role in transport to the
lysosome. Biochemical experiments indicated that particulate
Ykt6 in brain and neuroendocrine cells was not extractable
with salt or extremes of pH, but was fully extracted with
nonionic detergent. Likewise, particulate Ykt6 had a buoyant
density similar to lysosomal membranes. These features of
particulate Ykt6 suggested that the unique punctate staining
pattern probably represented a membrane compartment
(Hasegawa et al., 2003). Intriguingly, localization of
mammalian Ykt6 to the particulate structures did not require
prenylation. Instead, the N-terminal profilin-like domain of
Ykt6 was necessary and sufficient for normal localization
(Hasegawa et al., 2003).
Another striking feature of Ykt6 was its existence in both
particulate and freely soluble pools (Hasegawa et al., 2003).
Both yeast and mammalian Ykt6 lack transmembrane domains
but are prenylated at their C-termini (McNew et al., 1997).
Interestingly, the cytosolic pool of Ykt6 appears to be fully
prenylated in both species (Hasegawa et al., 2003; McNew et
al., 1997). This feature implied that Ykt6 must either have an
escort protein to sequester the lipid group(s), or else must
provide its own intramolecular lipid escort domain. One
possibility is that the Ykt6 profilin-like N-terminal domain
could provide the lipid escort function, in addition to mediating
the targeting to the Ykt6 membrane structures. This domain
might be involved in other regulatory features as well.
Cytosolic Ykt6 was found to be refractory to promiscuous
SNARE interactions, unlike bacterially expressed recombinant
Ykt6, suggesting the existence of an inhibitory regulatory
mechanism in the cytosol. Because Ykt6 behaved as a
monomer in solution, an intrinsic autoinhibitory mechanism
seemed more likely than regulation by other proteins.
Consistent with an intrinsic autoinhibited state, cytosolic Ykt6
was present in a more compact, protease resistant conformation
than the more promiscuous recombinant Ykt6. Because
SNARE N-terminal domains are established, in the case of
syntaxins, to fold back and pack against the SNARE motif and
inhibit SNARE complex formation (Munson et al., 2000), one
possibility was that the Ykt6 profilin-like domain did
something similar in the case of cytosolic, but not recombinant
Ykt6. In support of an autoregulatory role for this domain, the
yeast Ykt6p N-terminal domain was found to engage in
protein-protein interactions with the SNARE motif that caused
a modest inhibition in the kinetics of Ykt6p SNARE complex
assembly (Tochio et al., 2001).
The profilin-like domain of Ykt6 has been recognized to be
part of a larger family of domains with virtually identical
protein folds despite little or no sequence similarity. These
domains have been termed ‘longin’ domains, because they
account for certain R-SNAREs being longer than others
(Filippini et al., 2001). In mammals, three R-SNAREs contain
longin domains: Ykt6, Sec22b and VAMP7. The atomic
structure of the yeast Ykt6p (Tochio et al., 2001) and
mammalian Sec22b (Gonzalez et al., 2001) longin domains
have been solved and are virtually superimposable; the VAMP7
longin domain is assumed to have a similar profilin-like fold
and has also been implicated in protein localization (MartinezArca et al., 2003). Interestingly, similar domains appear to
reside on two non-SNARE isoforms of Sec22, called rSec22a
(Hay et al., 1996) and hSec22c (Tang et al., 1998). Likewise,
another non-SNARE protein, sedlin, whose structure was
recently determined to be superimposable with the SNARE
longin domains, appears to consist entirely of a longin domain
and nothing else (Jang et al., 2002). Although obviously not a
SNARE, sedlin is probably involved in ER-to-Golgi transport,
because it is the mammalian homolog of Trs20p, a subunit of
the TRAPP ER-Golgi tethering complex (Sacher and FerroNovick, 2001). Point mutations in human sedlin cause the
disease spondyloepiphyseal dysplasia tarda (Gedeon et al.,
1999). Longin domains appear to be versatile structures that
might regulate SNARE activity as well as perform other
essential roles in transport reactions.
We have taken advantage of the unique subcellular
localization of Ykt6 and the known structure of the Ykt6p
longin domain to define the structural features that control
Ykt6 targeting. We discovered that the Ykt6 longin domain
controls Ykt6 localization not only by direct targeting to the
Ykt6 subcellular particle but also by acting as a chaperone for
the Ykt6 SNARE motif and lipid group(s). In so doing, the
Ykt6 longin domain simultaneously keeps the fully lipidated
SNARE soluble, masks spurious targeting signals that would
otherwise cause abnormal localization and directs the molecule
to its specialized subcellular location.
Materials and Methods
Mammalian expression plasmids
All the cDNA sequences used in this study have been reported
previously: rat Ykt6 (GenBank accession number AA956066); yeast
Ykt6p (GenBank accession number NC001143); human sedlin
(SEDL) (GenBank accession number BC008889); mouse VAMP7
(GenBank accession number BC003764); rat Sec22a (GenBank
accession number NM057147); mouse Sec22b (GenBank accession
number U91538); rat GOS-28 (GenBank accession number U49099).
Various point mutants used in the targeting studies were generated by
polymerase chain reaction (PCR) based site-directed mutagenesis
using cDNAs that encode yeast Ykt6p longin domain (residues 1139), full-length yeast Ykt6p, rat Ykt6 longin domain (residues 1-137)
or full-length rat Ykt6 as templates. Truncation mutants for rSec22a,
mSec22b and mVAMP7 were created by introducing a stop codon
after residues 157, 134 and 125, respectively. All the chimeric proteins
used in this study were produced by gene splicing by overlap
extension (Horton et al., 1989). For the longin-domain-replacement
study, cDNAs that correspond to residues 1-157 of rSec22a, residues
1-134 of mSec22b, residues 1-125 of mVAMP7 or residues 1-140 of
hSEDL were directly spliced to cDNA that encoded residues 137-198
of rYkt6. For the transmembrane-domain-replacement study, cDNA
corresponding to residues 1-193 of rYkt6 was directly joined to
cDNAs corresponding to residues 196-215 of mSec22b, residues 187220 of mVAMP7 or residues 231-250 of GOS-28. Rat Ykt6 SNARE
motifs used in the binding assay were made by PCR amplification of
Ykt6 Targeting
cDNAs corresponding to residues 137-198 for lipidated SNARE motif
and residues 137-193 for non-lipidated SNARE motif. For N-terminal
Myc-tagging, all the DNA sequences described above, except for
SEDL-related sequences, were cloned into pCMV-Myc (Hay et al.,
1997) using SacII/XbaI sites. Full-length hSEDL was cloned into
pCMV-Tag3B (Stratagene) using BamHI/XhoI sites and the chimeric
cDNA encoding hSEDL(1-140)/rYkt6(137-198) was cloned into
BamHI/EcoRV sites of pCMV-Tag3B after 3′-end blunting with XbaI.
Expression plasmids coding hemagglutinin (HA) tagged yeast fulllength Ykt6p and yeast Ykt6p longin domain (residues 1-139) were
created by inserting corresponding PCR products into SacII/EcoRVdigested pCruzHA (Santa Cruz Biotechnology). For each construct,
the sequence was verified at the University of Michigan Sequencing
Core Facility by reading both strands.
Immunofluorescence microscopy
PC12 cells were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) containing 4.5 g/l glucose, 5% equine serum and 5% ironsupplemented calf serum (Hyclone, Logan, UT) without antibiotics in
a humidified 5% CO2 incubator at 37°C. PC12 cells plated on polylysine-coated glass coverslips in standard six-well culture dishes were
transfected using Lipofectamine 2000 (Invitrogen). Except where
methanol fixation was used, transfected cells were fixed in 0.1 M
sodium phosphate, pH 7.2, 2% paraformaldehyde and 0.05% Triton
X-100, for 30 minutes at room temperature 24-36 hours after
transfection. Fixed cells were then quenched twice in PBS containing
0.1 M glycine for 10 minutes each and incubated in permeabilization
buffer [0.4% saponin, 1% bovine serum albumin (BSA), 2% goat
serum in PBS] for 15 minutes at room temperature. This was followed
by incubation in primary antibodies for 1 hour in permeabilization
buffer. After four consecutive washes (5 minutes each) in
permeabilization buffer, cells were incubated with secondary
antibodies in permeabilization buffer for another 1 hour. Primary
antibodies used in this study were chicken anti-rat Ykt6 (Hasegawa
et al., 2003), mouse anti-Myc monoclonal antibody (hybridoma
9E10), rabbit polyclonal anti-Myc (Covance), mouse anti-HA
monoclonal antibody (hybridoma 16B12), rabbit anti-GM130 (gift
from M. Lowe, University of Manchester, UK), mouse monoclonal
anti-syntaxin 1 (hybridoma HPC-1) and anti-syntaxin 5 (Hay et al.,
1997). FITC- and Texas-Red-conjugated secondary antibodies were
purchased from Jackson ImmunoResearch. After the secondary
antibody labeling, cells were washed in permeabilization buffer four
more times and coverslips were mounted using Vectashield mounting
medium (Vector Laboratories), then sealed with nail polish.
LysoTracker Red DND-99 (Molecular Probes) was used to label
acidic compartments of PC12 cells. To inhibit in vivo palmitoylation,
PC12 cells were incubated in 150 µM 2-bromopalmitate (Aldrich),
starting 5 hours after transfection until cells were fixed at 30 hours
after transfection. These conditions are similar to those used in
previous studies (Webb et al., 2000).
Refer to Hasegawa et al. (Hasegawa et al., 2003) for detailed
description of epifluorescence microscopic image capturing and
image deconvolution. Quantification of colocalization in
immunofluorescence images was carried out using ImageJ software
(US National Institutes of Health) and calculated as the number of
doubly labeled pixels (positive for both Myc and endogenous Ykt6)
divided by the total number of Myc-positive pixels.
Bacterial expression of protein fusions with rat Ykt6 longin
domains
Wild-type rat Ykt6 longin domain (residues 1-137) was PCR
amplified and cloned into pGEX-KG (Guan and Dixon, 1991) using
XbaI/SacI restriction sites to make an N-terminal glutathione-Stransferase (GST) fusion protein. Using this wild-type rat Ykt6 longin
domain as template, mutant Ykt6 longin domains (V8D, F39E/F42E,
4497
R50E/R56E or V59E) were created by using the same PCR-based
site-directed mutagenesis used above. The sequence of all the mutants
in pGEX-KG plasmid were verified at the University of Michigan
Sequencing Core Facility by reading both strands. Expression
plasmids coding GST-rYkt6 longin wild-type and mutants were
transformed into Escherichia coli strain JM109. Transformants were
cultured in LB medium at 37°C. When the optical density at 600 nm
of the E. coli culture reached 0.5, cultures were shifted to 15°C and
protein production was induced by adding 0.1 mM IPTG for 6 hours.
Bacterial cultures was centrifuged at 6000 g and the pellet was
resuspended in French press buffer (50 mM Tris-HCl, pH 8.0, 0.1 M
NaCl, 1 mM EDTA, 0.05% Tween 20) supplemented with protease
inhibitor cocktail (Roche). After cell lysis by French press, the lysate
was first centrifuged at 20,000 g for 30 minutes and then at 100,000
g for 1 hour. The final supernatant was stored in aliquot at –80°C until
used in the binding assay.
Binding assay
For bead preparation, bacterial lysates containing wild-type or mutant
GST-rYkt6 longin domain were mixed with 10 µl bed volume of
glutathione/Sepharose-4B (Amersham Biosciences) in 0.5 ml
microcentrifuge tubes to immobilize GST fusion proteins. After an
overnight incubation at 4°C with constant rotation, the beads were
washed five times in binding buffer (20 mM Hepes, pH 7.2, 150 mM
KCl, 2 mM EDTA, 5% glycerol, 0.1% BSA, 0.1% Triton X-100).
Bound proteins were then analysed by sodium-dodecyl-sulfate
polyacrylamide-gel electrophoresis (SDS-PAGE) and Coomassieblue staining. Equivalent loading of six different proteins was
achieved by adjusting the input lysate.
To prepare the lipidated rYkt6 SNARE motif and non-lipidated
motif, confluent PC12 cell cultures in 10 cm dishes were transfected
with Myc-rYkt6 (residues 137-198) and Myc-rYkt6 (residues 137193), respectively. At 24 hours after transfection, the cells were
detached from the plates using Hanks’ balanced salt solution
containing 1 mM EDTA and collected by centrifugation. The cell
pellets were resuspended in lysis buffer [20 mM Hepes, pH 7.2, 250
mM KCl, 2 mM EDTA, 5% glycerol, 0.3% Triton X-100 (v/v),
protease inhibitor cocktail]. After a 30 minute incubation on ice,
lysates were centrifuged at 100,000 g for 1 hour and supernatant was
used as the ligand-containing fraction for the binding assay. To
determine the relative concentrations of the two different Myc
constructs, both lysates were subjected to SDS-PAGE (3-20%
gradient gel) and analysed by western blotting; the lipidated and nonlipidated recombinant SNARE motifs were detected by anti-Myc
(9E10) and anti-rat-Ykt6 antibodies as a band of the expected size (~8
kDa). For the bimolecular binding assay, a 10 µl bed volume of
protein-loaded glutathione/Sepharose-4B was mixed with 50-100 µl
PC12 cell lysate and binding buffer in a total assay volume of 150 µl.
The assay mixture was incubated at 4°C for 1 hour with constant
rotation. The beads were washed five times, then the beads were
boiled in 40 µl SDS sample buffer. For SDS-PAGE, 20 µl of each
sample was loaded on a 3-20% gradient gel. The proteins were
electrotransferred to nitrocellulose membranes, followed by
immunoblotting with anti-Ykt6 antisera.
SNARE complex assembly assay
ER/Golgi SNAREs (syntaxin 5, membrin and rBet1) were expressed
in bacteria as GST fusion proteins, and cleaved and purified using our
standard methods (Xu et al., 2000). Myc-rYkt6 (residues 137-193)
and Myc-rYkt6 (residues 137-198) were prepared as above. For
details of the SNARE assembly assay, refer to Hasegawa et al.
(Hasegawa et al., 2003). Briefly, PC12 extract containing Myc-rYkt6
137-193 was incubated overnight on ice in the presence or absence of
2 µM each of the ER/Golgi SNAREs in a total volume of 300 µl of
binding buffer The incubations were then gel filtered on a 24 ml
4498
Journal of Cell Science 117 (19)
Fig. 1. Space-filling model of Ykt6p longindomain structure. Surface-exposed residues
that are conserved from yeast to human are
highlighted with colors. White residues are
not conserved. Views of two sides of the
domain are shown. Structural coordinates are
obtained from the Protein Data Bank
(number 1H8M). Yellow indicates nonpolar
residues, green indicates polar residues, red
indicates acidic residues and blue indicates
basic residues. The figure was generated
using Deep View Swiss-PDB Viewer (The
Swiss Institute of Bioinformatics).
Superdex-200 column, and the gel filtration fractions analysed by
immunoblotting using the anti-Ykt6 antibody.
Membrane fractionation
PC12 cells were cultured in growth medium with or without 150
µM 2-bromopalmitate for 24 hours under normal culture
conditions. Cells were then harvested in Hank’s/EDTA buffer and
collected by centrifugation. Cell pellets were resuspended and
homogenized in homo buffer (20 mM HEPES, pH 7.0, 0.25 M
sucrose, 2 mM EGTA, 2 mM EDTA) supplemented with protease
inhibitor cocktail (Roche, Complete), followed by centrifugation
for 10 minutes at 1000 g to obtain a post-nuclear supernatant (PNS).
A portion of the PNS fraction was reserved for analysis and the
remainder was centrifuged at 100,000 g for 60 minutes to separate
the membrane and soluble fractions. All fractions were subjected
to acetone precipitation and the precipitated pellets were boiled in
SDS sample buffer. Equivalent amounts of each fraction were then
subjected to SDS-PAGE and immunoblotting with anti-GAP-43
antibody (hybridoma 9-1E12).
Preparation and immunohistochemistry of hippocampal
neuronal cultures
Primary hippocampal cultures containing both neurons and glial cells
were prepared from the CA1-CA3 regions (excluding the dentate
gyrus) of 1-4-day-old rats (Wistar; Taconic M&B, Ry, Denmark). The
hippocampi were dissected out and incubated in 1% trypsin (Sigma,
T1005) for 4-5 minutes at room temperature before they were
dissociated by triturating with silicone-coated Pasteur pipettes in the
presence of 0.05% DNase (Sigma, D5025). The cells were counted
and plated on Matrigel (Becton Dickinson, USA) coated small
coverslips (Assistent, Germany) at a density of ~92,000 cm–2. The
cells were allowed to attach by incubating them in a single drop for
15 minutes before more cell medium was added [Gibco’s modified
Eagle’s medium (MEM) with the addition of 0.3 mg ml–1 glutamine,
2.5 µg ml–1 insulin (Sigma, I-5500), 5-10% fetal calf serum (FCS), 2
µl ml–1 B-27 (Gibco) and 0.02-0.10 µl ml–1 ARA-C (Sigma, C6645)].
The cultures were maintained in a 5% CO2, 95% air incubator at 37°C.
Their medium was changed at 1 day, 4 days, 8 days and 12 days in
vitro (DIV). The medium at 1 DIV contained ARA-C to inhibit
cellular proliferation; 10% FCS was used at day 0 but, later, the added
medium contained 5% FCS. The cultures were used for experiments
at around 15 DIV.
Cultures at 15 DIV were fixed in 2% paraformaldehyde by the
following procedure. Freshly prepared fixative [2% paraformaldehyde
in 0.1 M sodium phosphate buffer (NaPi) pH 7.4] was heated to 37°C
before adding it to the culture medium (equal volumes). After 30
minutes, this mixture was substituted with fixative alone. After 1 hour,
the fixative was substituted by 0.1 M NaPi containing 0.2%
paraformaldehyde. The cells were quenched twice with 0.1 M glycine
in 0.1 M NaPi for 10 minutes, and subsequently permeabilized for 15
minutes in PS (0.4% saponin, 1% BSA and 2% normal sheep serum
in 0.1 M NaPi). The cultures were incubated in primary antibody in
PS for 1 hour, washed three times in PS alone before incubation in
secondary antibody in PS for 30 minutes, followed by one wash in PS
and three washes in 0.1 M NaPi. The coverslips were then mounted
with glycerol and subjected to investigation with a Leica TCS NT
confocal laser-scanning microscope. The chicken anti-Ykt6 antibody
was used at 1:10 and the anti-Sec6 antibody (9H5, hybridoma
supernatant) was undiluted. Secondary antibodies were RhodamineRed-X-conjugated anti-chicken and Cy2-conjugated anti-mouse
(Jackson ImmunoResearch, Baltimore, USA) used at 1:100.
Results
Longin-domain targeting to Ykt6 puncta is driven by its
conserved tertiary structure
A previous report demonstrated that sequence-specific
localization determinants reside within the mammalian Ykt6
longin domain and target the SNARE to an unidentified,
apparently membranous structure in PC12 pheochromacytoma
cells (Hasegawa et al., 2003). Because the yeast Ykt6p longin
domain can precisely target to the same vesicular structures in
PC12 cells, it seemed likely that conserved surface features of
the Ykt6 longin domain were responsible for targeting. We
examined the solved nuclear magnetic resonance (NMR)
structure (Tochio et al., 2001) of the yeast Ykt6p longin domain
for surface residues that were conserved between yeast and
mammals. Fig. 1 shows a space-filling model of the yeast Ykt6p
longin domain, highlighting the 31 conserved surface residues,
which are labeled and color coded as hydrophobic (yellow),
polar (green), acidic (red) or basic (blue). Mutations in 28 of
these residues were introduced, singly or in pairs, into the
isolated, Myc-tagged yeast longin domain and transfected into
PC12 cells. As shown in Fig. 2 and summarized in Table 1, none
of the surface mutations affected Ykt6p-longin-domain
targeting to the cytoplasmic Ykt6 puncta. Owing to space
limitations, only six mutants are shown in Fig. 2. Because the
yeast longin domain does not cross-react with our chicken antirat-Ykt6 antibody (Hasegawa et al., 2003), we were able to
detect both the Myc-tagged longin domains and the endogenous
rat Ykt6 to confirm that, in each case, the longin mutants
Ykt6 Targeting
Table 1. Localization of yeast Ykt6p mutants*
Localization phenotype
Surface 2
Surface 1
Surface 3
Mutation(s)
Ykt6p-longin
Ykt6p full-length
R2E/V8N
R2E/V8N/∆CCIIM
R2E
V8N
R71E
E100K/Y101N
E100K/Y101N/∆CCIIM
D99K
E100K
Y101N
Normal
–
ND
ND
Normal
Normal
–
ND
ND
ND
V113E/L120E
L120E/M122E
R88E
K131E/D134K
D139K
D24N/S26D
L25Q/F28Q
Normal
Normal
Normal
Normal
Normal
Normal
Normal
L25Q/F28Q/∆CCIIM
L25Q
F28Q
–
ND
ND
F30E/F31E
Normal
F30E/F31E/∆CCIIM
F30E
F31E
–
ND
ND
E32R/R33E
V36Q/Q38K
V36Q/Q38K/∆CCIIM
V36Q
Normal
Normal
–
ND
Q38K
ND
F39E
F39E/∆CCIIM
F42E
F42E/∆CCIIM
R50E/R56E
R50E/R56E/∆CCIIM
R50E
Normal
–
Normal
–
Normal
–
ND
R56E
ND
I59E
I59E/∆CCIIM
Normal
–
Plasma membrane
Normal
Normal
Plasma membrane
Normal
Plasma membrane
Normal
Normal
Normal
Spots+slightly highlighted
Golgi/plasma membrane
Normal
Normal
Normal
Normal
ND
Normal
Spots+slightly highlighted
Golgi/plasma membrane
Normal
Normal
Spots+slightly highlighted
Golgi/plasma membrane
Spots+slightly highlighted
Golgi/plasma membrane
Normal
Normal
Spots+slightlyhighlighted
Golgi/plasma membrane
Normal
Plasma membrane+Golgi
Normal
Spots+slightly highlighted
Golgi/plasma membrane
Spots+slightly highlighted
Golgi/plasma membrane
Plasma membrane+Golgi
Normal
Plasma membrane+Golgi
Normal
Plasma membrane+Golgi
Normal
Spots+slightly highlighted
Golgi/plasma membrane
Spots+slightly highlighted
Golgi/plasma membrane
Plasma membrane+Golgi
Normal
*ND indicates localization not determined. Mutants in bold are those
shown in Figs 2 and 4.
displayed a similar localization to rat Ykt6. Several
representative colocalizations are shown in Fig. 2. The longin
mutants often colocalized with endogenous Ykt6, producing a
total staining overlap of 12-18% (Fig. 2). We do not know why
many puncta were positive for the longin constructs
and negative for endogenous Ykt6, and vice versa. One
possibility is inefficient labeling with both antibodies caused by
epitope masking on the Ykt6 structures. The mutant longin
constructs colocalized with endogenous Ykt6 to the same
degree as full-length yeast Ykt6p constructs (Hasegawa et al.,
2003) [supplementary Fig. S1B (http://jcs.biologists.org/
supplemental/)]. This experiment indicated that conserved
sequence-specific surface features were probably not
4499
responsible for the ability of yeast and rat Ykt6 to target to the
same vesicular structures.
It should be realized that the localization of tagged Ykt6p
constructs and the degree of colocalization between these
constructs and endogenous Ykt6 were independent of the tag
used or the fixation method. Supplementary Fig. S1A
(http://jcs.biologists.org/supplemental/) demonstrates that
both HA/yeast-Ykt6p and HA/yeast-Ykt6p-longin-domain
displayed 17-18% colocalization with endogenous Ykt6, and
Fig. S1B demonstrates that Myc/yeast-Ykt6p displayed 24%
colocalization with endogenous Ykt6 in methanol-fixed PC12
cells.
Because mutation of surface-exposed residues did not
disrupt normal targeting, we wondered whether the overall
tertiary structure of the longin domain was responsible for the
targeting of the isolated longin domain to Ykt6 puncta. The
longin domain is constructed of a profilin-like protein fold that
is virtually superimposable between different proteins having
the domain, despite limited or no sequence similarity (Dietrich
et al., 2003). We expressed the isolated Myc-tagged longin
domains from the SNAREs VAMP7 and mSec22b, the nonSNARE Sec22 homolog rSec22a, and sedlin [a protein
composed of only a longin domain and that has been
implicated in vesicle tethering reactions and the genetic disease
spondyloepiphyseal dysplasia tarda (Jang et al., 2002)]. As
shown in Fig. 3A, all of these Myc-tagged longin domains
targeted the cytoplasmic puncta in PC12 cells and overlapped
with endogenous Ykt6 to a similar degree as bona fide Ykt6p
constructs (Fig. 3). This implies that it was actually the
profilin-like structural scaffold rather than sequence-specific
surface features of Ykt6 that are responsible for targeting the
isolated domain. It is important to realize that VAMP7 and
Sec22b isoforms do not normally colocalize with Ykt6 in PC12
cells (not shown), presumably because the full-length proteins
possess other targeting determinants. How can the experiment
in Fig. 3A, implicating the overall longin structure and not
Ykt6-specific features, be reconciled with the previous
observation that normal Ykt6 targeting required sequencespecific features of the Ykt6 longin domain (Hasegawa et al.,
2003)? As shown in Fig. 3B, when chimeras containing each
of the above longin domains spliced onto the SNARE motif
and C-terminus of Ykt6 were expressed in PC12 cells, all of
them displayed generalized mislocalization to the Golgi area
and plasma membrane. Thus, although any longin domain on
its own can target to the Ykt6 punctate structures, only the Ykt6
longin domain is sufficient to target the whole Ykt6 molecule
to these cytoplasmic structures. This suggested that the Ykt6
longin domain might have sequence-specific surface features
that suppress otherwise-dominant ‘mistargeting’ determinants
present on the SNARE motif and/or prenylation motif.
Sequence-specific surface features of the Ykt6 longin
domain regulate targeting
To examine the possibility of longin-domain surface features
that suppress otherwise-dominant targeting determinants on the
Ykt6 SNARE motif and prenylation site, we introduced the
same panel of surface mutations examined above into fulllength Myc-tagged yeast Ykt6p and expressed them in PC12
cells. As shown in Fig. 4 and summarized in Table 1, several
surface mutations resulted in striking mislocalization
4500
Journal of Cell Science 117 (19)
Fig. 2. Mutant Ykt6p longin domains are
targeted normally. Mutations were
introduced into N-terminally Myc-tagged
yeast Ykt6p longin domain (residues 1-139)
and transfected into PC12 cells. See Table 1
for the complete listing of mutations.
Subcellular localization of each mutant and
endogenous rat ykt6 was examined by
double-labeling fluorescent microscopy
using anti-Myc antibody (9E10) and antirat-Ykt6 antibody. Whereas colocalization
of mutants with endogenous rat Ykt6 is
shown for R2E/V8N, F39E and I59EA (top
three rows; 12%, 18% and 16% overlap,
respectively), only the subcellular
localization of Myc-tagged mutant proteins
is shown for E100K/Y101N, R50E/R56E
and F42 (bottom row). Arrows point to a
subset of overlapping punctate structures.
Scale bar, 10 µm.
phenotypes. In all cases, double-labeling indicated that the
localization of endogenous Ykt6 was unaffected. The surface
residues that affected Ykt6 localization fell into three groups
that might define distinct surfaces important in the control of
Ykt6 targeting. The first surface is composed of a conserved
hydrophobic patch including F39, F42, V36 and the flanking
residue Q38. Of these residues, F39 and F42 had been
previously shown to participate in intramolecular interactions
between the Ykt6p longin domain and the SNARE motif. These
intramolecular interactions appeared to exert a weak inhibitory
effect on SNARE-SNARE interactions using purified,
nonprenylated, recombinant SNAREs (Tochio et al., 2001). A
second apparent surface was composed of a mixture of charged
residues and exposed hydrophobic residues, including R50, R56
and I59, on the same face of the domain as the above
hydrophobic patch. These residues have not been previously
implicated in intramolecular interactions. A third surface
consisted of a deep, conserved hydrophobic crevice on the
opposite face of the longin domain and included V8 and Y101,
and the flanking charged residue E100. Again, these residues
were not observed to interact with the SNARE motif in the
previous NMR studies with purified, nonprenylated Ykt6p. All
of the aforementioned mutations on the first and second surfaces
resulted in mislocalizations that included Golgi area as well as
apparent plasma membrane staining, with a few cytoplasmic
vesicles that did not discernibly colocalize with endogenous
Ykt6 (Fig. 4, F39E, F42E, I59E,
R50E/R56E). Interestingly, mutations in
the hydrophobic crevice (surface 3)
resulted in predominantly plasma
membrane staining and overlapped by
>45% with syntaxin-1 staining (Fig. 4,
V8N, E100K/Y101N). None of the
surface mutations examined in Fig. 4 are
likely to have caused major disruptions
of the tertiary structure of the longin
domain because, when present in the
isolated longin domain, they did not
detectably affect localization or stability
of the transfected proteins (Fig. 2). The
results of Fig. 4 indicate that
intramolecular interactions might be important for control of the
normal localization of Ykt6 in PC12 cells.
Mammalian Ykt6 targeting is regulated by similar
surface features of the longin domain
All of the preceding work was done using yeast Ykt6p and the
yeast Ykt6p longin domain. To determine whether similar
intramolecular interactions control the localization of the
mammalian Ykt6, we repeated most of the analysis using the
bona fide mammalian protein that is enriched in brain neurons
and PC12 cells. As shown in Fig. 5 and Table 2, all three of the
surfaces identified in the yeast protein similarly affected the
localization of the mammalian Ykt6, although there were a few
residues in mammalian Ykt6 that had less severe mislocalization
phenotypes. Similar to what we found for the yeast protein, none
of the localization-disrupting mutations had any effect on the
localization or expression level of the isolated longin domain,
implying that it was probably surface features and intramolecular
interactions rather than overall structural disruptions that resulted
in the mislocalization phenotypes with the full-length constructs.
Longin domain suppresses promiscuous targeting
signals in the Ykt6 C-terminus
We wondered which structural features of Ykt6 were
Ykt6 Targeting
4501
Fig. 3. Normal targeting of Ykt6
requires both conserved and Ykt6specific targeting signals in the longin
domain. (A) Several distinct longin
domains, when removed from their
normal protein context, can localize to
the Ykt6 vesicular structures. Longin
(lgn) domains from Sec22b, VAMP7,
Sec22a and sedlin were N-terminally
Myc-tagged and expressed in PC12
cells. Subcellular localization of each
Myc-longin domain and endogenous rat
Ykt6 was examined by double-label
fluorescence microscopy using anti-Myc
antibody (9E10) and anti-rat-Ykt6
antibody. In each row, the left-hand
panel shows the localization of a longin
domain of different origin, the middle
panel shows endogenous Ykt6 and the
right-hand panel is the merged image.
Arrows point to punctate structures
where the Myc-longin domain and
endogenous rat Ykt6 colocalize (22%,
19%, 22% and 14% overlap,
respectively). Scale bars, 10 µm.
(B) Only the Ykt6 longin domain can
direct normal subcellular targeting of
the Ykt6 SNARE and prenylation
motifs. The longin domain of rat Ykt6
was replaced by exogenous longin
domains to create N-terminally Myctagged chimeric Ykt6 proteins. Each
chimeric rat Ykt6 was expressed in
PC12 cells and the subcellular
localization was examined by
fluorescent microscopy using anti-Myc
antibody and FITC-labeled secondary
antibody. Scale bars, 10 µm.
responsible for the abnormal targeting of the protein observed
in the various mutants described above. As shown in Fig.
6 and summarized in Tables 1 and 2, deletion of the Cterminal CCAIM sequence (CCIIM in yeast), which is the
prenylation motif, rescued the abnormal localization
phenotypes of all yeast and mammalian mislocalization
mutants, restoring cytoplasmic vesicular punctate staining
that displayed normal amounts of overlap with endogenous
Ykt6. For reasons of space, Fig. 6 shows only a handful of
examples. Formally, hydrophobic modifications at the Cterminus of Ykt6 present dominant mistargeting information
that is normally masked by sequence-specific surface features
of the longin domain.
Hydrophobic crevice including V8 might interact with a
palmitoyl group on C194
We wondered whether any of the longin domain surfaces were
specifically involved in suppressing otherwise-dominant
determinants associated with one or the other of the pair of
cysteines at the Ykt6 C-terminus. The precise structure of the
fully modified Ykt6 terminus is not known in yeast or
mammals. The sequence would predict farnesylation (a C15
isoprenoid), although geranylgeranylation is also a possibility.
Probably, the second cysteine (C195) is linked to a farnesyl
lipid. The upstream cysteine (C194) could be unmodified or
be farnesylated or palmitoylated. Other farnesylated proteins
with a double cysteine motif (e.g. Ras) are palmitoylated on
4502
Journal of Cell Science 117 (19)
Fig. 4. Surface mutations on the Ykt6p
longin domain disrupt the targeting of fulllength Ykt6p. The same complete set of
mutations (Fig. 2, Table 1) were introduced
into N-terminally Myc-tagged yeast fulllength Ykt6p. Mutants were expressed in
PC12 cells as before. Localization of each
mutant was examined by co-staining with
anti-rat-Ykt6 or anti-syntaxin-1 antibodies.
(Top row) The subcellular localization of
Myc-tagged yeast Ykt6p mutant proteins
E100K/Y101N, F42E and R50E/R56E.
(Second row) The localization of the V8N
mutant stained with anti-Myc (polyclonal)
and anti-syntaxin 1 (47% overlap)
antibodies. (Third and fourth rows) Double
labeling of mutant-construct-expressing
cells with anti-Myc (E10) and anti-Ykt6
antibodies. These constructs displayed 1.6%
and 2.6% overlap with endogenous Ykt6,
respectively. In the second case, the bright
perinuclear area of the mutant was excluded
from the calculation owing to obviously
coincidental overlap. Scale bar, 10 µm.
the upstream cysteine (Hancock et al., 1989). In these cases,
palmitoylation of the first cysteine requires prior farnesylation
of the second cysteine. Hence, based upon Ras, it would not
be feasible to delete the second cysteine to create a
palmitoylated but not farnesylated mutant. However, it would
be possible, by mutation of the upstream cysteine, to create a
farnesylated protein that does not get palmitoylated. As shown
in Fig. 6B and Table 2, mutation of C195 to an alanine restored
the collection of mammalian mislocalization mutants to
normal distribution, just like deletion of the entire motif had
done, consistent with a lack of modification of either cysteine.
However, mutation of C194 to alanine had various effects on
the mislocalized constructs. For mutations of the large
hydrophobic patch (surface 1, including F39 and F42), which
had previously been implicated in protein-protein interactions
with the SNARE motif, and the mixed hydrophobic/charged
surface on the same face (surface 2, including V59), the
substitution restored some normal, spotty cytoplasmic
Table 2. Localization of rat ykt6 mutants*
Longin mutations
Longin only
Full-length
None
V8D
E100K/F101N
V112E
I118E/Y120E
F30E/F31E
F39E/F42E
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Plasma membrane and Golgi
Normal
Normal
Normal
Normal
Plasma membrane and Golgi
R50E/R56E
V59E
Normal
Normal
Plasma membrane and Golgi
Plasma membrane and Golgi
Full-length+
C194A
Full-length+
C195A
Full-length+
C194A/C195A
Full-length+
∆CCAIM
Normal
Normal
ND
ND
ND
ND
Spots+slightly highlighted
Golgi/plasma membrane
ND
Spots+slightly highlighted
Golgi/plasma membrane
Normal
Normal
ND
ND
ND
ND
Normal
Normal
Normal
ND
ND
ND
ND
Normal
Normal
Normal
ND
ND
ND
ND
Normal
ND
Normal
ND
Normal
ND
Normal
*ND indicates localization not determined. Mutants in bold are those shown in Fig. 5 and Fig. 6B.
Ykt6 Targeting
4503
Fig. 5. Rat Ykt6 localization is
regulated by similar longin-domain
surface features. Mutations to the longin
domain were introduced to full-length
rat Ykt6 and truncated rat Ykt6
composed of only a longin domain
(residues 1-137). All the mutants were
expressed in PC12 cells and the
subcellular localization of each mutant
was examined by fluorescent
microscopy using anti-Myc antibody
(9E10) and FITC-labeled secondary
antibody. (Top) Mutants in the fulllength context. (Bottom) Mutants in the
isolated Ykt6 longin constructs. Scale
bar, 10 µm.
staining, but there remained significant abnormal staining in
the secretory pathway and plasma membrane areas. This is
consistent with the hypothesis that Ykt6 is indeed modified on
both cysteines and with these two longin-domain surfaces
being partially responsible for suppression of targeting
information from the upstream cysteine. However, targeting of
the V8D mislocalization mutant was seemingly completely
restored to normal Ykt6 staining by substitution of C194
only. This implies that this hydrophobic crevice might be
specifically involved in masking the hydrophobic moiety
attached to C194.
Interestingly, unlike other mutants, mislocalization mutants
in the V8 crevice, especially of the yeast Yk6p (Fig. 4), were
predominantly mislocalized to the plasma membrane only,
implying release of only a portion of the targeting determinants
masked by the longin domain. Because palmitoylation is known
to target proteins specifically to the plasma membrane and
palmitoylation is generally found on the upstream cysteine in
farnesylated proteins with double cysteines, our results imply
that the crevice around V8 might be a binding pocket for a
palmitoyl group attached to C194. In support of this hypothesis,
as shown in Fig. 7A, incubation of cells overnight with
2-bromopalmitate, a competitive inhibitor of protein
palmitoylation, restored at least the normal level of
colocalization to two distinct mutations affecting the V8
hydrophobic surface on yeast Ykt6p (V8N, K100E/Y101N)
while having no discernible effect on mutants of surface 1
(F42E or F39E) or surface 2 (not shown). The 2bromopalmitate conditions used did, in fact, abrogate protein
palmitoylation, because GAP-43 (a plasma membrane
palmitoyl protein present in neurons and PC12 cells) underwent
a drastic reduction in membrane association upon the drug
treatment (Fig. 7B).
The simplest interpretation of the data in Fig. 6B and Fig. 7
is that Ykt6 is palmitoylated on C194 and the palmitoyl group
is normally sequestered in the longin-domain hydrophobic
crevice around V8. However, no direct demonstration of Ykt6
palmitoylation has been reported, and other models are
possible. For example, the hydrophobic crevice could interact
with palmitoyl groups on other proteins that are affected by
bromopalmitate treatment. Yeast Ykt6p has recently been
demonstrated to participate in a specific protein-palmitoylation
event (Dietrich, 2004).
Longin-domain mislocalization mutants disrupt proteinprotein and protein-lipid intramolecular interactions
The evidence presented so far suggests that intramolecular
protein-protein and protein-lipid interactions might control the
targeting of Ykt6 in neuroendocrine cells. One way in which
this might be accomplished would be the formation of a
compact conformation in which the longin domain doubles
back and packs against the SNARE motif, and the lipid
moieties protrude back along the SNARE motif to be buried in
binding sites on the longin-domain surface. To test whether the
localization phenotypes we observed are indeed due to the
disruption of such a conformation of Ykt6, we developed a
bimolecular binding assay between purified, recombinant rat
Ykt6 longin domain on beads and soluble longin-domaindeleted Ykt6 constructs present in detergent extracts of PC12
cells. As shown in Fig. 8A, a specific interaction was observed
between the GST/longin-domain and the longin-deleted Ykt6
construct with an intact CCAIM motif. This binding
phenomenon was not complicated or affected by the inclusion
of the construct in Triton X-100 micelles, because the same
result was obtained using octyl-glucopyranoside extracts
diluted well below the critical micellar concentration
[supplementary
Fig.
S2
(http://jcs.biologists.org/
supplemental/)]. Strikingly, every mutation we tested that had
disrupted the localization of full-length Ykt6, from each of the
three surfaces, completely abrogated binding between the
respective GST/longin-domain and the soluble longin-deleted
Ykt6 construct containing CCAIM. This implies that, as we
hypothesized, the three highlighted surface features are indeed
4504
Journal of Cell Science 117 (19)
Fig. 6. Mistargeting is rescued by
removal of C-terminal lipids.
(A) Effect of C-terminal deletions on
yeast Ykt6p mislocalization mutants.
The last five amino acid residues
(CCIIM) were deleted from yeast
Ykt6p mislocalization mutants and
expressed in PC12 cells (Table 1).
The subcellular localization of each
mutant and endogenous rat Ykt6 was
examined by double-label
fluorescence microscopy using antiMyc antibody (9E10) and anti-ratYkt6 antibody. (Top left) Myc-tagged
yeast Ykt6p F39E with CCIIM
deletion. (Bottom left) R50E/R56E
with CCIIM deletion. Arrows point
to punctate structures where the
mutants and endogenous rat Ykt6
colocalize (overlaps were 31% and
18%, respectively). Identical results
were obtained when CCIIM was
deleted from other mislocalization
mutants (Table 1). Scale bar, 10 µm.
(B) Effect of C-terminal mutations
on rat-Ykt6 mislocalization mutants.
Rat Ykt6 mutants that have
mutations in both the longin domain
and the C-terminal lipidation motif
were created (Table 2). Mutants were
expressed in PC12 cells and the
subcellular localization of each
mutant was examined by
fluorescence microscopy using antiMyc antibody and FITC-labeled
secondary antibody. Mutations to the
longin domain are indicated at the
left and mutations to the C-terminus
are indicated at the top. Scale bar,
10 µm.
important for intramolecular interactions, resulting in a Ykt6
conformation that sequesters spurious targeting signals on the
SNARE motif and/or modified C-terminus. Although surface
residues in the first of the three highlighted surfaces had been
previously implicated in protein-protein interactions between
the longin domain and SNARE motif, we wondered whether
any of the identified intramolecular interactions were in fact of
a protein-lipid nature. Deletion of the CCAIM modification site
at the C-terminus of the longin-deleted construct completely
eliminated all detectable binding with GST-longin protein (Fig.
8A, lower blot). The Myc-rYkt6 137-193 construct was in fact
functional/accessible for protein interactions in the extract,
because incubation of the cell extract with purified ER/Golgi
SNAREs resulted in quantitative incorporation of the construct
into high-molecular-weight SNARE complex(es) detectable by
gel filtration (Fig. 8D). In addition, purified rYkt6 137-198
produced in bacteria (and thus not lipidated) failed to interact
detectably with GST-longin beads in the same binding assay
format (not shown). The results of Fig. 8 imply that, indeed,
longin-domain/lipid interactions contribute to the normal
targeting of the molecule to cytoplasmic structures, probably
by formation of a compact conformation that sequesters the
SNARE motif and C-terminal lipids.
Normal Ykt6 targeting requires a soluble intermediate
If Ykt6 indeed functions as a SNARE on the vesicular
structures, one might wonder why this SNARE would rely
upon a soluble intermediate for targeting to this site of
function. Prenyl proteins are thought to be initially inserted into
the endoplasmic reticulum during the modification process
(Gelb et al., 1998), so it would be conceivable for Ykt6 to travel
Ykt6 Targeting
4505
Fig. 7. 2-Bromopalmitate suppresses the
mislocalization phenotype of selected
mutants. (A) PC12 cells were transfected
with full-length yeast Ykt6p mutants. At 5
hours after transfection, the growth
medium was changed to a medium
containing 2-bromopalmitate and incubated
until the cells were fixed. After fixation,
subcellular localization of each mutant and
endogenous rat Ykt6 was examined by
double-labeling fluorescent microscopy
using anti-Myc antibody and anti-Ykt6
antibody. Top panels show co-staining of
endogenous Ykt6 (left) and Myc-Ykt6pV8N (middle), and merged (right) images
after drug treatment. Arrows point to
puncta where colocalization is observed
(27% overlap). The same degree of
colocalization was observed for MycYkt6p-K100E/Y101N (bottom left) and
endogenous Ykt6 (not shown). Mislocalization phenotypes of MycYkt6p-F42E (bottom center), Myc-Ykt6p F39E (bottom right), MycYkt6p R50E/R56E (not shown) and Myc-Ykt6p I59E (not shown)
were not altered by the 2-bromopalmitate treatment. Scale bar,
10 µm. (B) GAP-43 translocates from membrane to cytosol in the
presence of 2-bromopalmitate. Localization of endogenous GAP-43
was analysed by membrane fractionation followed by
immunoblotting. Abbreviations: P, pellet fraction; S, soluble fraction;
T, total fraction. Presence or absence of 2-bromopalmitate (2-BP) is
indicated at the top.
to its destination compartment using the endomembrane
system after insertion into the ER, like other SNAREs. To test
whether the Ykt6 longin domain could provide the targeting
information necessary to target the SNARE to the Ykt6
structures through the endomembrane system, we removed the
lipid modification site from the full-length wild-type Myctagged construct and replaced it with each of several
proteinaceous transmembrane domains (TM) taken from the
late endosomal SNARE VAMP7, the ER/Golgi SNARE
Sec22b and the medial/trans-Golgi SNARE GOS-28. As
shown in Fig. 9, the Ykt6 longin domain was completely
unable to target the transmembrane Ykt6 constructs to its
normal vesicular structures. In fact, in each case, the construct
localized similarly to the SNARE from which the
transmembrane domain was derived; Myc-Ykt6/Sec22bTM
localized to the Golgi area and colocalized with syntaxin 5,
Myc-Ykt6/VAMP7TM localized to the limiting membrane of
abnormally swollen, acidic lysosome-like structures and MycYkt6/GOS28TM localized to the Golgi area but did not
colocalize well with the cis-Golgi marker GM130, consistent
with the construct being localized to the late Golgi. We
conclude that either the normal punctate Ykt6 structure is
unavailable from the mainstream endomembrane system or
the targeting determinants in the Ykt6 longin domain are
subordinate to the signals present in the transmembrane
domains used. In either case, it emphasizes the importance of
the intramolecular interactions described above for the ‘proper’
subcellular targeting of Ykt6 in cultured neuroendocrine cells.
It was interesting that the Myc-Ykt6/VAMP7TM construct
caused aberrant lysosome-like structures (Fig. 9, middle). This
implies that Ykt6 might have a role in lysosome fusion,
consistent with the function of Ykt6p in yeast. However, it does
not clarify the relationship between the unique Ykt6 punctate
structures and lysosomes and/or Ykt6 function.
Ykt6 subcellular targeting in primary hippocampal
neurons
The endogenous punctate Ykt6 structures do not colocalize
significantly with markers of the endomembrane system
(Hasegawa et al., 2003), so we considered the possibility that
they were not a natural structure but an aberrant structure
present only in transformed cell lines like PC12. We thus
investigated the subcellular localization of endogenous and
Myc-tagged recombinant Ykt6 expressed in primary cultures
of hippocampal neurons. As shown in Fig. 10A, endogenous
Ykt6 staining (red) was punctate in nature, similar to that in
PC12 cells and ng108 cell lines. Punctate staining was heavy
throughout the entire cell body of neurons, as well as major
dendrites and axons. The Ykt6 structures did not appear to be
enriched in any particular part of the cell but rather
accumulated wherever there was a significant volume of
cytoplasm. As shown in Fig. 10B-E, Ykt6 puncta were even
observed within individual synaptic terminals, where the
presynaptic plasma membrane was indicated with an antibody
to the exocyst component rSec6. This work demonstrates that
the unusually specialized localization of Ykt6 in cultured
neuroendocrine cells is similar to that observed in bona fide
brain neurons, where Ykt6 is most abundant (Hasegawa et al.,
2003).
4506
Journal of Cell Science 117 (19)
Fig. 8. Localization mutants display disrupted protein-protein and
protein-lipid intramolecular interactions. (A) Results of bimolecular
binding assay. Bead-immobilized GST-longin domains were mixed
with a Triton-X-100 cell lysate prepared from PC12 cells
overexpressing either lipidated (Myc-rYkt6 residues 137-198) or
non-lipidated (Myc-rYkt6 residues 137-193) SNARE motif and
incubated at 4°C for 1 hour. Beads were washed, boiled in SDS
sample buffer, subjected to SDS-PAGE and immunoblotted to detect
the bound SNARE motif. The lipidated (residues 137-198) and nonlipidated (residues 137-193) Myc-SNARE motifs are indicated by
arrows. (B) Equivalent protein loading to the beads. Proteins bound
to 1.25 µl bed volume of glutathione-Sepharose beads were resolved
on a 15% gel by SDS-PAGE and stained with Coomassie blue.
(C) Equivalent amount of SNARE motif was used for the binding
assay. 10% of the PC12 extracts containing the SNARE motifs used
in the binding assay were analysed by SDS-PAGE and
immunoblotting. (D) Myc-rYkt6-137-193 actively assembles with
ER/Golgi SNAREs. Cell lysate containing Myc-rYkt6-137-193 was
incubated with buffer alone or with purified, recombinant, ER/Golgi
SNAREs (2 µM each), followed by Superose-12 gel filtration
chromatography. Column fractions were immunoblotted with antiYkt6 antibody. Elution positions of molecular weight standards are
indicated above.
Discussion
Many features of mammalian Ykt6 remain a mystery. Perhaps
most importantly, we do not yet know the molecular identity
of the punctate structures to which the protein targets or
whether Ykt6 functions there as a SNARE. Markers of the
endomembrane system did not significantly colocalize with
Ykt6, but particulate Ykt6 behaved as a membrane-associated
protein with respect to detergent extractability and buoyant
density. By contrast, cytosolic Ykt6 behaved as a purely
monomeric protein, without a hint of aggregation or higherorder organization (Hasegawa et al., 2003). The best
interpretation of the data so far is that cytosolic Ykt6 is an
intermediate in the localization to the specialized compartment
(i.e. that cytosolic Ykt6 targets to the punctate structures by
binding to them directly from the
cytosol). Whether the punctate
structures
represent
traditional
membrane-bilayer-enclosed organelles
is still an active question.
We have now thoroughly defined the
structural features of Ykt6 that allow
targeting to its unique membrane
location. The overall conserved longin
tertiary structure, rather than sequencespecific or surface structure, contains
Fig. 9. Transmembrane-anchored Ykt6
constructs cannot localize normally to
punctate Ykt6 structures. The C-terminal
lipidation motif (CCAIM) of rat Ykt6 was
replaced with the true transmembrane
domain (TM) of Sec22b, VAMP7 or GOS28. The chimeric proteins were expressed in
PC12 cells and their subcellular
localizations were examined by
fluorescence microscopy using anti-Myc
antibody and the indicated co-stained
marker. Arrows point to gigantic aberrant
membrane structures with acidic lumens
induced by Ykt6 containing a VAMP7
transmembrane domain. Scale bar, 10 µm.
Ykt6 Targeting
4507
ykt6
Lgn
targeting
component
ykt6 punctate structure
Fig. 10. Ykt6 exhibits a very broad vesicular cytoplasmic
localization in cultured hippocampal neurons. Hippocampal neurons
and glia from 1-4-day-old rats were cultured for 15 days in vitro
before fixation and staining with anti-rat-Ykt6 (red) and anti-Sec6
(green) antibodies, and analysed by confocal microscopy.
(B-E) Individual synaptic terminals with the plasma membrane
clearly demarcated by Sec6 staining. Scale bar, 25 µm (A), 2 µm (E).
the signal for direct localization to the Ykt6 compartment.
However, the Ykt6 longin domain indirectly controls the
localization of the protein by regulating the availability
of targeting signals in the SNARE motif and C-terminal
lipid moieties. This regulation involves sequence-specific
intramolecular interactions between the longin domain and the
C-terminal lipids and SNARE motif. We identified three
distinct surfaces on the Ykt6 longin domain that are important
for these intramolecular interactions. One included residues
previously identified by Banfield and colleagues to participate
in protein-protein interaction with the SNARE motif (Tochio
et al., 2001). Another surface on the same face of the domain
appears to involve a mixture of hydrophobic and charged
residues. Finally, a third surface represents a deep hydrophobic
crevice on the opposite face of the domain. Indirect evidence
we present suggests that this crevice might represent a
palmitoyl-group binding pocket. Our interpretation of the role
of the intramolecular interactions in targeting is that they allow
the SNARE to adopt a compact, closed conformation that
sequesters the SNARE motif and lipid(s), thus preventing
otherwise promiscuous targeting interactions.
Interestingly, significant bimolecular interactions were not
observed between the longin domain and SNARE motif of
Ykt6 in the absence of lipid modification (Fig. 8A). At first
glance, this seems at odds with Banfield and colleagues, who
reported that intramolecular protein-protein interactions
between the longin domain and SNARE motif were detectable
by NMR in the purified, recombinant, non-hydrophobically
modified Ykt6 (Tochio et al., 2001). However, those purely
protein-protein interactions might have been weak or transient,
as indicated by their very modest inhibitory effect on SNARE
complex formation. Our interpretation of the results is that the
lipid group(s)
Fig. 11. Model for Ykt6 intramolecular interactions (left), targeting
(middle) and activation upon membrane insertion (right). In this
model, the Ykt6 longin domain (Lgn) interacts with the lipid groups
(black) and the SNARE motif (red) such that the lipids are shielded
from solvent and from premature insertion into membranes and the
SNARE motif is sequestered and unavailable to other SNAREs.
However, the longin domain is still available for interaction with a
targeting component on the Ykt6 compartment. This allows inactive
Ykt6 to be targeted to its specialized location, where binding
between the targeting component and the longin domain results in:
(1) interaction of the lipid group(s) with the membrane; (2) release of
the longin domain from its interaction with the C-terminal lipid(s);
and (3) release of the Ykt6 SNARE motif from sequestration to allow
potential SNARE interactions.
protein-protein interactions by themselves are not strong
enough to be detected in the bimolecular binding assay we
used. However, they contributed significantly, because
mutation of the residues (e.g. F42) that Banfield and colleagues
found to be important for protein-protein interactions (Tochio
et al., 2001) inhibited binding in our assay. Thus, we propose
that both protein-protein and protein-lipid interactions are
required to produce a stable intramolecular interaction and
tightly closed conformation.
Our present and previous (Hasegawa et al., 2003) results
suggest several unique conformational properties of Ykt6 in
neuroendocrine cells, which are summarized in the model of
Fig. 11. As depicted, fully prenylated, perhaps palmitoylated
Ykt6, exists as a freely soluble monomer before targeting to
its specialized vesicular structure. It maintains solubility by
having the longin domain act as a lipid chaperone that
sequesters these groups in hydrophobic binding pockets.
Partly as a consequence of the intramolecular protein-lipid
interactions, the longin domain is locked in place tightly
against the SNARE motif, which also interacts with the longin
domain. This compact cytosolic conformation sequesters
would-be promiscuous interactions of the SNARE motif and
lipid group(s), thus preventing mistargeting. The longin
domain of cytosolic Ykt6 is, however, free to interact with an
unknown targeting component on the particulate Ykt6
compartment. This targeting interaction requires only the
tertiary structure of the longin domain and is not directly
affected by any of the surface mutations explored in this study.
Once targeted to the Ykt6 particle, the lipid group(s) might
4508
Journal of Cell Science 117 (19)
partition into a fully lipidic environment, strongly anchoring
Ykt6 there in a detergent-extractable but not chaotropeextractable condition. The retraction of the lipid group(s) from
their longin-domain-binding sites would dramatically weaken
the longin-SNARE intramolecular interactions, opening up
the molecule and making the SNARE motif available for
potential participation in SNARE interactions. The existence
of intramolecular protein-protein as well as protein-lipid
interactions allows the longin domain to regulate Ykt6
conformation over a wide dynamic range, such that it virtually
shuts off all interactions when soluble, whereas only mildly
modulating SNARE availability when embedded in
membranes. Hopefully, future work will elucidate the nature
of the particulate structure that neuronal Ykt6 goes to such
lengths to arrive at.
This work was supported by NIH grant MH 68524 to J.C.H. S.D.
and L.O. received support from EU grant QLG3-CT-2001-02089.
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