From the Russell Grimwade School of Biochemistry & Molecular Biology, University of Melbourne, Parkville 3010, Australia,
the § Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, and Department of Cancer Biology,
The Dana-Farber Cancer Institute, Boston, Massachusetts 02115, and
the ¶ Bioinformatics Group, MEMOREC Stoffel GmbH, Stoeckheimer Weg
1, D-50829 Köln, Germany
Received for publication, December 13, 2002, and in revised form, January 3, 2003
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ABSTRACT |
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Tail-anchored proteins have an
NH2-terminal cytosolic domain anchored to
intracellular membranes by a single, COOH-terminal, transmembrane
segment. Sequence analysis identified 55 tail-anchored proteins in
Saccharomyces cerevisiae, with several novel proteins, including Prm3, which we find is required for karyogamy and is tail-anchored in the nuclear envelope. A total of six tail-anchored proteins are present in the mitochondrial outer membrane and have relatively hydrophilic transmembrane segments that serve as targeting signals. The rest, by far the majority, localize via a bipartite system
of signals: uniformly hydrophobic tail anchors are first inserted into
the endoplasmic reticulum, and additional segments within the cytosolic
domain of each protein can dictate subsequent sorting to a precise
destination within the cell.
Tail-anchored proteins have a single transmembrane segment at
their carboxyl terminus, and many of the proteins that mediate subcellular traffic and programmed cell death are tail-anchored into
select membranes of eukaryotic cells (1). In the Bcl-2 family of
proteins, key regulators of the programmed cell death pathway in animal
cells, 12 of the 16 known family members are tail-anchored to either
the mitochondrial outer membrane or endoplasmic reticulum, and their
membrane location is critical for function (2). The
SNAP-receptors
(SNAREs)1 are a family of
proteins essential for intracellular membrane fusion, and 19 of the 23 SNAREs in yeast are tail-anchored proteins. Membrane fusion absolutely
requires the participation of SNAREs in both the donor and acceptor
membrane, and each of the known SNAREs has a restricted location at a
defined membrane compartment of the endomembrane system (3, 4).
Tail-anchored proteins fold co-translationally and the single
hydrophobic segment at their carboxyl terminus allows for
post-translational insertion into membranes (5, 6). Once membrane is
inserted, the amino-terminal domain of the protein is displayed in the
cytosol. An elegant study on the tail-anchored SNARE
synaptobrevin-1/VAMP-1a found that the protein is inserted into the
endoplasmic reticulum and subsequently sorted to presynaptic vesicles
(5). Cytochrome b5, another tail-anchored
protein, is inserted into the membrane of the endoplasmic reticulum and
maintained there despite some escape to, and retrieval from, the
cis-Golgi cisternae (7).
But tail-anchored proteins are also located in the mitochondrial outer
membrane, and the precise signal that distinguishes these from those
targeted to the endoplasmic reticulum is still not clear. Deletion
mutagenesis has shown the signal is contained within the tail segment,
and in the few different model proteins examined to date critical
determinants have been either the presence of charged residues or in
some cases the number of hydrophobic residues (8-14). An understanding
of the precise targeting signals that direct the majority of
tail-anchored proteins to the endoplasmic reticulum, but allow some to
go exclusively to mitochondria, has been hampered by the relatively
small number of model tail-anchored proteins that have been available
for study.
We applied several bioinformatic approaches to identify
tail-anchored proteins encoded in the genome of
Saccharomyces cerevisiae. Fifteen novel
tail-anchored proteins were discovered; we report here on the
localization of the previously unrecognized tail-anchored proteins.
Analysis of the targeting segments from the 55 tail-anchored proteins
in yeast suggests a bipartite system of signals: hydrophobic character
in the tail segment determines targeting to the endoplasmic reticulum
instead of mitochondria and discrete sorting signals that then direct
tail-anchored proteins to their correct subcellular destination.
Plasmids and Yeast Strains--
DNA fragments corresponding to
each open reading frame were amplified by PCR using primers that
generated one in-frame restriction site immediately preceding the start
codon and another following the stop codon (oligonucleotide sequences
available on request). PCR products were cloned behind GFP-S65T
under the control of the MET25 promoter and expressed from a
centromeric plasmid (13). In semisynthetic (SD) media, expression from
the plasmid is partially repressed.
PCR-based mutagenesis was used to convert hydrophilic residues in the
transmembrane segments of Tom22 and Fis1 to leucine residues. For the
mutant described here, Fis1(4L), Gly136,
Gly137, Gly141, and Ala142 were
converted to leucine residues. Initial trials to visualize expression
of the fusion proteins were made in the diploid strain JK9-3da/ Fluorescence Microscopy--
For fluorescence microscopy, cells
were visualized directly or after staining with Mitotracker
(MitoTracker Red CM-H2X Ros) according to the standard protocol from
Molecular Probes. All fluorescence images were captured using a Bio-Rad
MRC1024 confocal scanning laser microscope mounted on a Zeiss Axioscop.
For the mating studies, a Nikon fluorescence microscope with GFP and
DAPI filter sets and a ×100 DIC (Nomarski) objective was used. In this case images were captured by a Micromax digital camera with Metamorph imaging software. In preparation for fluorescence microscopy cells were
grown to mid-log phase at 25 °C in semisynthetic medium. In
assays for nuclear transport, wild-type, rna1-1, or
prp20-1 cells were transferred from 25 °C liquid culture
to a 37 °C water bath for 30 min immediately prior to preparation
for microscopy.
Membrane Isolation and Analysis--
Microsomal membrane
fractions were prepared by differential centrifugation. Cells (50-100
OD600 units) were suspended in buffer 88 (250 mM sorbitol, 150 mM potassium acetate, 5 mM magnesium acetate, 0.5 mM
phenylmethylsulfonyl fluoride, 1.2 µg/ml leupeptin, 0.75 µg/ml antipain, 0.25 µg/ml chymostatin, 1 µg/ml pepstatin, 50 mM HEPES, pH 6.8) and disrupted by two bursts (each of
2-min duration) in a mini-beadbeater-8 (Biospec products) using
silica/zirconia beads. Cell debris was removed by centrifugation at
500 × g for 5 min. A crude membrane fraction was
collected by centrifugation at 16,000 × g for 10 min.
Membranes were extracted by resuspension in either 1% Triton X-100 or
100 mM Na2CO3 and incubated
for 30 min on ice with intermittent vortexing. Soluble and insoluble proteins were separated by centrifugation at 100,000 × g in a Beckman Airfuge.
Miscellaneous--
Published procedures were used for isolation
of mitochondria and trypsin shaving, SDS-PAGE, and immunoblot analysis
(16). Detailed comparative hydrophobicity analyses of the targeting sequences in each tail-anchored protein made use of the ProtParam site
at (expasy.proteome.org.au/cgi-bin/protparam) using a window of
5 amino acids to scan through the predicted transmembrane segment according to the Kyte-Doolittle algorithm.
In addition to the tail-anchored proteins known in yeast, sequence
analysis revealed 15 open reading frames that could be expressed as GFP
fusions that localize to discrete subcellular membranes. Fig.
1 shows that Fis1 and YFL046w are
mitochondrial proteins (Fig. 1A), seven proteins localized
generally to the endoplasmic reticulum membrane (Fig. 1B),
and four are localized to specific subdomains of the endoplasmic
reticulum (Fig. 1C). YLR238w and YOR324c are found in
clusters within the bounds of the endoplasmic reticulum, and Prm3 is
confined to the perinuclear (nuclear envelope) membrane. YPL206c, a
protein showing sequence similarity to bacterial glycerophosphodiester
phosphodiesterases, was found concentrated in lipid bodies, regions of
endoplasmic reticulum specialized for lipid metabolism (Fig.
1C; Ref. 17). Two proteins closely related in primary
structure, YDL012c and YBR016w, are located in the plasma membrane
(Fig. 1D). Both YDL012c and YBR016w are localized more
intensely in regions of new membrane synthesis, toward the emergent
buds of dividing cells and also in the schmoo structure (Fig.
1E, arrowheads) of haploid cells in the presence
of mating pheromone.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(leu2-3,122/leu2-3,122,
ura3-52/ura3-52, rme1/rme1
trp1/trp1, his4/his4 GAL+/GAL+, HMLa/HMLa).
In three cases, this failed to give discernible fluorescence but
expression of fusion proteins constructed from YBL100c, YFL046w, and
YPL200c was possible using a strain defective in proteasome function
(Mat
, trp1, ura3, his, leu2, cim5-1; Ghislain
et al. (38)). To generate yeast mutants lacking the
FIS1 gene or the PRM3 gene, PCR-mediated gene
disruption (15) was employed with the plasmid p3xHA-His5 as template.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Localization of tail-anchored proteins
in vivo. A, yeast cells expressing the
Fis1 and YFL046w fusions were co-stained with the fluorescent dye
Mitotracker and viewed by fluorescence microscopy. Filters selective
for the green fluorescence of GFP (left section of each
panel) or the red fluorescence of Mitotracker (right section
of each panel) were used. B, each of the indicated open
reading frames targets GFP to the endoplasmic reticulum
(arrows, peripheral membrane; N denotes nucleus
and perinuclear membrane). C, YLR238w and YOR324c show a
punctate staining pattern, Prm3 displays restricted perinuclear
localization, and YDR200c is enriched in lipid bodies. D,
YDL012c and YBR016w target GFP to the plasma membrane where the
proteins are enriched in new buds. E, in haploid
(Mata) cells incubated for 2 h in the
presence of untransformed cells of the opposite mating type (Mat )
and visualized by fluorescence microscopy, YBR016w is enriched
in mating projections of "schmoo" cells
(arrowheads).
The Tail Segment Is Necessary for Targeting to the Endoplasmic
Reticulum--
Comparative sequence analysis of the carboxyl-terminal
segments of the 41 tail-anchored proteins targeted to the endoplasmic reticulum (including the 17 proteins sorted to other membranes of the
secretory pathway) revealed no obvious motifs in the primary structure
that might serve as a common targeting signal. However, hydropathy
analysis through the transmembrane segments suggests regions of high
(>3.3 units) hydropathy score in each protein (Fig.
2A). Conversely, hydropathy
analysis of the predicted transmembrane domain from Fis1 (Fig.
2B) and for YFL046w, Tom5, Tom6, Tom7, and Tom22 (data not
shown) suggests this segment of each polypeptide is more amphipathic
than for proteins targeted to the endoplasmic reticulum. Previous work
has shown that the transmembrane segments of the translocase subunits
Tom5, Tom6, Tom7, Tom22, and Fis1 are necessary and sufficient for
targeting mitochondria (13, 14,
18).2
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To test whether the character in the transmembrane segment of Fis1 distinguished it as a protein destined for mitochondria, site-directed mutagenesis was used to replace hydrophilic residues with leucines, such that the hydrophobicity approached that of proteins targeted to the endoplasmic reticulum (Fig. 2B). When yeast cells expressing these mutant proteins as GFP fusions were analyzed by fluorescence microscopy, the Fis1 mutant, Fis1(L4), is targeted to the endoplasmic reticulum (Fig. 2C). Similar results were found with mutations made in the transmembrane segment of Tom22 (data not shown). Fluorescence is sometimes also observed in the lumen of the vacuole, perhaps reflecting turnover of the inappropriately targeted proteins (Fig. 2C, "V").
Bipartite Signals for Targeting and Sorting Proteins in
Intracellular Membranes--
Tail-anchored proteins in the endoplasmic
reticulum can display distinct patterns of localization. For example
YOR324c is found in discrete clusters throughout the endoplasmic
reticulum (Fig. 3A), whereas
Bos1 is a SNARE found sparingly in the endoplasmic reticulum but
enriched in the Golgi (Fig. 3B, arrows). The
hydrophobic tail segments from each of these proteins are sufficient to
target GFP to the endoplasmic reticulum, where it remains generally
distributed (Fig. 3, C and D). The proteins are
integrated into the membrane as judged by their resistance to
extraction by sodium carbonate treatment of isolated membranes (Fig.
3E; data not shown).
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We identified a single protein, Prm3, whose distribution was restricted
to the nuclear envelope and sought to understand whether its targeting
and sorting were also mediated by bipartite signals. A series of
deletion mutants were constructed (Fig.
4A) and tested for subcellular
localization. Deletion of 91 amino acids, leaving only the
carboxyl-terminal segment of Prm3 (GFP-Prm31-91), targets the GFP
reporter to the endoplasmic reticulum (Fig. 4B), which is
continuous with the outer membrane of the nuclear envelope (19). A
second signal is present in Prm3(
1-68), causing the fusion to be
localized specifically to the nuclear envelope (Fig. 4C).
Sequence analysis suggested a nuclear
localization sequence (NLS) between
Pro68 and Lys75.
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Point mutations in the putative NLS caused degradation of the
full-length Prm3 fusion protein, and so to test whether the putative
NLS is functional, we analyzed a soluble version,
GFP-Prm3(109-133). GFP-Prm3(
109-133) is localized exclusively
within the nucleus, and confocal sectioning revealed it distributed
throughout the nucleoplasm (Fig. 4D). To be certain the
information for nuclear localization is contained within the amino acid
sequence P68GRVRKHK75, site-directed
mutagenesis was used to convert Lys73 to Ala73,
and the mutant protein GFP-Prm3(
109-133)* is compromised in import,
with much of the protein located in the cytosol (Fig. 4E).
If Prm3 has a bipartite targeting sequence, with the P68GRVRKHK75 truly acting as an NLS, the general nuclear import machinery of the cell should be required to maintain the full-length, membrane-embedded Prm3 within the nucleus. Ran is a small GTP-binding protein that plays a critical role in transport of proteins into the nucleus (20, 21). A gradient of Ran-GTP across the nuclear membrane is established by the differential localization of two enzymes: the Ran-GTP exchange factor (Ran-GEF) in the nucleus and the Ran-GTP-activating protein (Ran-GAP) in the cytosol, and mutations in either of these two enzymes leads to collapse of nuclear traffic.
Temperature-sensitive mutants for Ran-GEF (prp20-1) and Ran-GAP (ran1-1) were transformed and the distribution of Prm3 measured by fluorescence microscopy. At the permissive temperature of 25 °C, the localization of Prm3 in the Ran-GEF mutants (Fig. 4F) and the Ran-GAP mutants (Fig. 4G) is nuclear. However, after shift to the non-permissive temperature of 37 °C, Prm3 is found throughout the membranes of the endoplasmic reticulum, suggesting that without a functioning Ran cycle the protein cannot be sorted from the outer to inner membrane of the nuclear envelope.
Since Prm3 is the first protein described in yeast that might be
localized to the inner membrane of the nuclear envelope, we sought to
determine its function by analysis of prm3 mutant cells.
Haploid yeast cells of opposite mating types, either wild type
(Mata) with wild type (Mat
) or
prm3
(Mata) with
prm3 (Mat
), were mixed and
incubated on rich medium for 3-4 h, then fixed and stained with
DAPI to visualize DNA. In wild-type crosses, nuclear DNA is usually
mixed by the time the first diploid bud appears near the junction of
the two parent cells of the mating pair (Figs.
5A). However, the cytoductants
that arise from
prm3 ×
prm3 crosses
are unable to undergo normal karyogamy, and nuclear material remains
distinct but tightly juxtaposed at the point where the nuclei
simultaneously squeeze through the bud neck, whether staining for
nucleic acid (Fig. 5B) or nucleoplasmic protein (Fig.
5C).
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If, as suggested by this microscopy, Prm3 is required for
the fusion of nuclear envelopes, then prm3 cells should
remain haploid through subsequent generations. To test this
possibility, ten zygotes from mating mixtures like those described in
Fig. 5 were isolated by micromanipulation. The progeny from wild-type crosses are diploid: in the course of mitosis they develop bud scars at
each end of the ovoid cells (Fig. 5D), they cannot mate with
haploid cells of either mating type, and they will undergo meiosis if
placed on sporulation media (data not shown). By contrast, the progeny
from
prm3 ×
prm3 matings were
exclusively haploid: the cells that arise mitotically from these
cytoductants have a single, spiralled pattern of bud scars (Fig.
5D), cannot be sporulated, but can mate with (wild-type)
cells of the opposite mating type (data not shown). The colonies that
initially arise from these cytoductants show a distinct furrow, with
cells isolated from one side of the furrow exhibiting mating type
a and cells from the other side being of mating type
.3
Transcript profiling has shown that the PRM3 gene is induced
in response to both pheromone stimulation (22) and during sporulation (23), and both processes culminate in karyogamy (see review by Rose
(24)). Because Prm3 localization is dependent on a NLS and the
Ran-GTPase cycle, we propose Prm3 as the first component of the
cellular karyogamy machinery to function in membrane fusion at the
level of the inner membrane of the nuclear envelope.
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DISCUSSION |
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In addition to the discovery and localization of a large set of novel tail-anchored proteins, analysis of the sequence data provided by these proteins suggests a general model for the targeting of tail-anchored proteins to each intracellular membrane.
From analysis of the 41 tail-anchored proteins now known to localize to membranes of the secretory pathway, the only common property we could identify was the uniformly hydrophobic nature of the residues within the transmembrane segment. Previous studies on individual tail-anchored proteins have each suggested that the tail-segment contains targeting information (8-14). The hydrophobic tail segments of Bos1, Prm3, and YOR324c are sufficient to target each of these proteins to the endoplasmic reticulum, and these truncated fusions remain uniformly distributed through the perinuclear endoplasmic reticulum (i.e. the outer membrane of the nuclear envelope) and peripheral endoplasmic reticulum.
A segment of lower hydrophobic moment, containing glycine, serine, and threonine residues, is found in the six tail-anchored proteins targeted to the mitochondrial outer membrane. Replacement of these residues with leucines, to alter the hydrophobicity and perhaps other structural features such as the rigidity of the helix it can form, prevents targeting to mitochondria. The TOM translocation machinery mediates protein insertion into the outer membrane (25-27), as well as the import of soluble proteins into mitochondria (28-31). The recent three-dimensional structure of the TOM complex receptor subunit, Tom20, suggests that it has only a very shallow binding groove on its surface, into which binds the hydrophobic face of the targeting sequences found attached to soluble proteins targeted to mitochondria (32, 33). Tom20 has been implicated as the import receptor for tail-anchored proteins, and it will be of interest to determine the structural details of how the receptor binds the targeting segments of tail-anchored proteins.
In contrast to insertion of tail-anchored proteins into mitochondria, the nature of the machinery that mediates tail-anchored protein insertion into the endoplasmic reticulum is still unclear. Kutay et al. (5) suggested that the general post-translational Sec machinery might mediate insertion of tail-anchored proteins like VAMP-1a. Whatever the machinery, it is able to insert tail-anchored proteins with hydrophobic tail-segments; even a simple, artificial tail-segment consisting entirely of leucine residues was a suitable substrate for in vitro insertion into membrane vesicles derived from the endoplasmic reticulum (34), and the same tail segment directs GFP to the endoplasmic reticulum when expressed in yeast cells.3
Distinct regions in the cytosolic domain of VAMP-1a assist its
subsequent transport from the endoplasmic reticulum to the presynaptic
vesicles. Interactions mediated by distinct regions in the cytosolic
domains are also required for Bos1 trafficking to the Golgi, Prm3
targeting to the nuclear envelope, and YOR324c clustering in punctate
zones of the endoplasmic reticulum. Thus two independent processes,
targeting to the endoplasmic reticulum and subsequent sorting to the
correct membrane, are mediated by bipartite targeting and sorting
signals. For the Golgi and post-Golgi membranes, sorting would be
mediated through vesicular traffic. In the case of Prm3 and any other
proteins that might exist in the inner membrane of the nuclear
envelope, we suggest that this occurs via the lipid rivulets of pore
membrane present in the nuclear pore complex (35, 36) that would link
the outer and inner membrane bilayers and is catalyzed by Ran and the
karyopherins that also drive the import of soluble nuclear proteins.
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ACKNOWLEDGEMENTS |
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We thank Ben Glick, Tina Junne-Bieri, and Jeff Schatz for plasmids and antisera; Peter Walsh, Diana Macasev, and Ross Waller for critical suggestions on the manuscript; and Binks Wattenberg and David Huang for comments on the manuscript and critical discussions throughout the course of the project.
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FOOTNOTES |
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* This work was supported by a grant from the Australian Research Council (to T. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Russell Grimwade
School of Biochemistry and Molecular Biology, University of Melbourne, Parkville 3010, Australia. Tel.: 61-3-8344-4131; Fax: 61-3-9348-2251; E-mail: t.lithgow@unimelb.edu.au.
Published, JBC Papers in Press, January 3, 2003, DOI 10.1074/jbc.M212725200
2 T. Beilharz and T. Lithgow, unpublished observations.
3 T. Beilharz, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors; DAPI, 4',6-diamidino-2-phenylindole; NLS, nuclear localization sequence; GEF, GTP exchange factor; GAP, GTP-activating protein.
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