* Laboratory of Cell Biology, Howard Hughes Medical Institute; and Laboratory of Cellular and Structural Biology, The
Rockefeller University, New York, New York 10021
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Abstract |
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While much has been learned in recent years about the movement of soluble transport factors across the nuclear pore complex (NPC), comparatively little is known about intranuclear trafficking. We isolated the previously identified Saccharomyces protein Mlp1p (myosin-like protein) by an assay designed to find nuclear envelope (NE) associated proteins that are not nucleoporins. We localized both Mlp1p and a closely related protein that we termed Mlp2p to filamentous structures stretching from the nucleoplasmic face of the NE into the nucleoplasm, similar to the homologous vertebrate and Drosophila Tpr proteins. Mlp1p can be imported into the nucleus by virtue of a nuclear localization sequence (NLS) within its COOH-terminal domain. Overexpression experiments indicate that Mlp1p can form large structures within the nucleus which exclude chromatin but appear highly permeable to proteins. Remarkably, cells harboring a double deletion of MLP1 and MLP2 were viable, although they showed a slower net rate of active nuclear import and faster passive efflux of a reporter protein. Our data indicate that the Tpr homologues are not merely NPC-associated proteins but that they can be part of NPC-independent, peripheral intranuclear structures. In addition, we suggest that the Tpr filaments could provide chromatin-free conduits or tracks to guide the efficient translocation of macromolecules between the nucleoplasm and the NPC.
Key words: nucleocytoplasmic transport; nucleoskeleton; nuclear pore complex; nuclear envelope; Saccharomyces cerevisiae ![]() |
Introduction |
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THE nuclear envelope (NE)1 defines the boundary
between the nucleus and the cytoplasm in eukaryotic cells. The NE is composed of two distinct but
continuous membranes enclosing a lumen. The outer nuclear membrane is continuous with the ER membranes
and is thought to perform rough ER functions (Fawcett,
1966; Baba and Osumi, 1987; Preuss et al., 1991
; Strambio-de-Castillia et al., 1995
). Facing the nucleoplasm is the inner nuclear membrane, which in multicellular eukaryotes
is often lined by a filamentous network called the nuclear
lamina (Gerace et al., 1978
; for review see Moir et al.,
1995
). The inner and the outer nuclear membranes join to
form specialized circular apertures of ~100 nm diameter containing the nuclear pore complexes (NPCs). NPCs provide the only known pathway for the exchange of cellular
material across the NE. The core of the NPC consists of a
cylindrical assembly of eight identical spoke structures symmetrically arranged around the central transporter (Unwin and Milligan, 1982
; Hinshaw et al., 1992
; Akey and
Radermacher, 1993
; Yang et al., 1998
). Images of the transporter suggest that it is a gated structure with a roughly
cylindrical shape (Akey, 1990
; Akey and Radermacher,
1993
; Goldberg and Allen, 1996
). Peripherally associated
nuclear and cytoplasmic filaments project from the core
(Feldherr et al., 1984
; Dworetzky and Feldherr, 1988
;
Akey and Goldfarb, 1989
). The nuclear filaments conjoin
to form the nuclear fishtrap or nuclear basket (Ris, 1991
;
Goldberg and Allen, 1992
). Transport substrates dock to
these filaments and translocate through the transporter on
their way in and out of the nucleus. The NPCs of Saccharomyces share many features with their vertebrate counterparts, though they are significantly smaller both in mass
and in volume (Yang et al., 1998
). A variety of immunological, biochemical and genetic techniques have been
successfully employed in the past few years to identify
NPC component proteins (nucleoporins). Three criteria
are commonly used to demonstrate that a novel protein is
a bona fide nucleoporin: the protein should (a) immunolocalize to the NPC by immunofluorescence (IF) microscopy
or better by immunoelectron microscopy (IEM); (b) cofractionate with the NPC in subcellular fractionation procedures; and (c) interact genetically and/or biochemically
with other known nucleoporins. To date 28 yeast proteins
and 15 vertebrate proteins have been identified that meet
at least two of the above mentioned criteria for a nucleoporin (for review see Rout and Wente, 1994
; Bastos et
al., 1995
; Doye and Hurt, 1997
; Fabre and Hurt, 1997
).
While it is clear that the NPC is a major regulator of nucleocytoplasmic transport, the knowledge of how this essential superstructure is connected both structurally and
functionally to the nuclear interior is still extremely limited. Recently, the investigation of the molecular basis of
such connections has received great impetus from the
study of vertebrate Tpr (translocated promoter region)
and of the Tpr-related Drosophila protein Bx34. The NH2-terminal ~200 amino acid residues of human Tpr have
been detected in various human tumors fused with the kinase domains of the three protooncogenes met, trk, and raf
(Park et al., 1986; Soman et al., 1991
; Greco et al., 1992
;
Mitchell and Cooper, 1992
). The sequence of Tpr predicts
a large protein (~265 kD) with a bipartite secondary structure (Byrd et al., 1994
; Bangs et al., 1996
). The NH2-terminal 70% of the polypeptide sequence (~184 kD) has
a high
-helical content and is predicted to give rise to a
coiled-coil domain. The remaining 30% of the protein
(~81 kD) is predicted to be acidic. While Tpr had been
initially localized exclusively to the cytoplasmic filaments
associated with the NPC (Byrd et al., 1994
), more recent work has conclusively demonstrated that Tpr is a constitutive component of long nuclear filaments (up to ~300 nm
in Xenopus) that appear to connect the distal ring of the
nuclear basket with the nucleolus. These filaments could
correspond to the 5-6-nm filaments described on numerous occasions (Franke, 1970
; Franke and Scheer, 1970a
,b;
Kartenbeck et al., 1971
; Richardson et al., 1988
; Ris and
Malecki, 1993
; Cordes et al., 1997
) and recently found to
form extensive networks of branching hollow cables that project from the nuclear baskets towards the nucleoplasm
in amphibian oocytes (Ris, 1997
). The predicted filamentous structure of Tpr together with its localization led to
the proposal that this protein could have a role in providing a structural framework for the transport of material
from the NPCs towards the interior and vice versa. This
conclusion has received support from results obtained with a Tpr-related protein in Drosophila, called Bx34. This
protein was initially described as one of two novel classes
of Drosophila NE antigens that had been identified using
mAbs obtained against chromosomal protein fractions
(Frasch et al., 1988
). Strikingly, Bx34 was recently found
both at the nuclear periphery in association or near NPCs
and in the nuclear interior in extrachromosomal and extranucleolar regions reminiscent of the extrachromosomal
channel network described in relation to mRNA processing and transport (Zimowska et al., 1997
). Consistent with
its localization pattern, Bx34 was found to cofractionate
exclusively with biochemical preparations of the nuclear
matrix suggesting that it could represent a component of
the nuclear matrix filamentous network. Interestingly,
Bx34 retains its association with the chromosomes until
very late in mitosis leading to the suggestion that it could
have a structural role in aiding chromosomal segregation
in anaphase. Further support for the involvement of Tpr in
nuclear transport has recently come from a report indicating that this protein can be coimmunoprecipitated from
Xenopus egg extracts together with karyopherins
and
1 (Shah et al., 1998
).
The yeast Saccharomyces presents certain advantages
over multicellular eukaryotes as a system to study such
proteins. They do not have the complications of nuclear
disassembly, having a closed mitosis; in addition, the genetics and molecular biology of yeast are better understood than in any other eukaryote, and the DNA sequence
of the entire yeast genome is now known (Clayton et al.,
1997). We describe here work aimed at isolating and characterizing non-NPC NE-associated proteins. Isolated NEs
prepared as previously described (Strambio-de-Castillia et
al., 1995
) were used to produce a panel of mAbs against
NE-associated components. A novel NPC-clustering assay
was devised to specifically isolate anti-NE mAbs that recognized antigens only partially or peripherally associated
with the NPCs. Two mAbs were isolated that recognized
an ~220-kD NE antigen that clustered only partially with
the NPCs in a NPC-clustering strain. The gene encoding
this antigen was molecularly cloned and was found to correspond to the previously isolated myosin-like protein (MLP1; Kolling et al., 1993
). This gene encodes a nuclear
protein of unknown function that is the closest yeast relative of Tpr. Ultrastructural localization and studies of deletion mutants strongly argues that Mlp1p and its homologue Mlp2p could be involved in providing a structural
and functional link between the NPCs and the nuclear interior.
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Materials and Methods |
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Subcellular Fractionation
The yeast strains Saccharomyces uvarum (NCYC 74, ATCC 9080), considered a strain of Saccharomyces cerevisiae (Mortimer and Johnson,
1986), or S. cerevisiae (W303; Thomas and Rothstein, 1989
) were used
throughout the procedure. Enriched nuclei, NEs and heparin-extracted
NEs were prepared as previously described (Rout and Kilmartin, 1990
,
1994
; Strambio-de-Castillia et al., 1995
; Rout and Strambio-de-Castillia,
1998
). Enriched NPCs were prepared from nuclei (fraction 7) exactly as
described in Rout and Blobel (1993)
and Rout and Strambio-de-Castillia
(1998)
.
Proteins contained in 30 ml of the highly enriched NE fraction described above (fraction 10), were precipitated by mixing the sample with 9 vol of methanol and harvested by centrifugation. The methanol pellet was
solubilized in 4 ml of 10 mM MES, pH 6.5, 100 mM DTT, 1% SDS at 90°C
for 10 min. The resuspended proteins were mixed with 36 ml of 20 mM
MES, pH 6.5, 7 M Urea, 1% (vol/vol) Triton X-100, 0.1% SDS, 1 mM
DTT (buffer 7). 16 ml of a 1:1 suspension of the cation-exchange S-Sepharose resin (8 ml of resin bed) were loaded on a broad base, 50-ml column
and washed three times with 20 ml of buffer 7. The NE sample was loaded
onto the column and was allowed to absorb onto the resin by incubating
for 1 h at 25°C with gentle rocking. After the binding step, the flow-through from the column was harvested and pooled with a 20-ml wash in buffer 7 (this pooled material was termed unbound fraction). The column
was eluted two times with 30 ml each of 1 M NaCl in buffer 7. Proteins
from both bound and unbound fractions were harvested by methanol precipitation. Aliquots were separated on SDS-PAGE. After electrophoresis,
the fractionation pattern of known NPC components was analyzed by immunoblotting using mAb414 (Davis and Blobel, 1986). The unbound fraction (termed S-NE), was found to be selectively depleted of most nucleoporins recognized by mAb414. Proteins from this fraction were
harvested by methanol precipitation, resuspended in PBS, and used to immunize mice.
Mice Immunization and Production of mAbs
The production of hybridomas from B-lymphocytes derived from mice
spleens was as previously described (Galfre and Milstein, 1981; Rout and
Kilmartin, 1990
). Supernatants were screened by indirect IF microscopy
of whole yeast cells (Rout and Kilmartin, 1990
; Wente et al., 1992
; Kilmartin et al., 1993
). Positive supernatants were also screened by immunoblotting of enriched NEs. Cells from positive lines were cloned up to four
times by limiting dilution using a standard protocol (Galfre and Milstein,
1981
; Rout and Kilmartin, 1990
).
Molecular Cloning and Sequence Analysis
A gt11 S. cerevisiae genomic expression library (Clontech Laboratories
Inc., Palo Alto, CA) was screened by immunoblotting using mAb148G11
following the specifications of the manufacturer. Three positive
clones
containing an identical insert of ~1.8 kb were obtained and purified to homogeneity by four consecutive rounds of screening. The insert from one
of the positive
clones was subcloned into pBluescript SK(+/
), sequenced from both ends using the T3 and the T7 standard primers and
found to contain a sequence identical to the coding region for AA 1,105-
1,700 of MLP1 (Myosin Like Protein 1; Kolling et al., 1993
). DNA sequence comparisons were performed using the BLAST algorithm (Altschul et al., 1990
). Deducted amino acid sequences were compared with sequences in the SGD (Saccharomyces Genome Database), GenBank and
EMBL databases using the FASTA algorithm (Parson and Lipman, 1988).
Amino acid sequence alignments were performed using FASTA and
CLUSTAL W v. 1.6 (Higgins et al., 1994
). Amino acid sequences were analyzed using Protean v. 1.08 (DNAStar Inc.) and MacStripe 1.3.1 (Lupas
et al., 1991
).
Ultrastructural Studies
IEM analysis of isolated NEs was performed using a modification of a
published procedure (Wray and Sealock, 1984; Rout and Kilmartin, 1990
;
Kraemer et al., 1995
). For the IEM of isolated whole nuclei, the nuclear
preparations were subjected to mild osmotic shock by diluting them with 9 vol of PVP solution (8% polyvinylpyrrolidone [PVP]; 20 mM potassium
phosphate, pH 6.5; 0.75 mM MgCl2). These broken nuclei were then transferred (100 µl of diluted sample per well) to microtiter wells pretreated as
described for the IEM of isolated NEs, and centrifuged at 23,500 g for 30 min at 4°C. Nuclei pellets were washed twice with PVP solution at 25°C
followed by one wash each for 5 min with the following three solutions:
(a) 25% (vol/vol) M buffer (5% dried milk in bt-DMSO [10 mM Bis-Tris-Cl, pH 6.50, 0.1 mM MgCl2, 20% DMSO]), 75% (vol/vol) PVP solution;
(b) 50% (vol/vol) M buffer, 50% (vol/vol) PVP solution; and (c) 75% (vol/
vol) M buffer, and 25% (vol/vol) PVP solution. The nuclei pellets were
washed once in M buffer for 5 min at 25°C. Subsequent steps were also as
described above with the following exceptions: (a) 0.5× PBS-K, 1 mM
MgCl2 was substituted with bt-DMSO; and (b) after treatment with osmic acid, samples were postfixed with 1% tannic acid in 50 mM potassium phosphate, pH 7.0, for 30 min at 25°C.
Localization of Green Fluorescent Protein Fusion Proteins
To generate green fluorescent protein (GFP)-tagged proteins we used
the plasmid pGFP-N-FUS encoding the GFP gene under the control of
the MET25 promoter (Niedenthal et al., 1996). DNA sequences encoding the
following Mlp1p fragments: (a) NT1 (AA 1-667; pGFPNT1); (b) NT2
(AA 668-1446; pGFPNT2); (c) CT (AA 1,447-1,875; pGFPCT); and (d)
putative nuclear localization sequence (NLS), pNLS (AA 1,486-1,545;
pGFPpNLS), were PCR-amplified and cloned in-frame downstream of
the GFP gene using the unique cloning sites XbaI and XmaI. pGFPNT1,
pGFPNT2, pGFPCT, pGFPpNLS, and the negative control pGFP-N-FUS
were transformed into W303 cells. To induce the expression of the GFP-tagged Mlp1p fragments, cells were grown to mid-log phase in selective
medium without methionine. GFP fusion proteins were visualized in living
cells after mounting on microscope slides with 2% methyl cellulose, using
a Zeiss Axiophot microscope. (Carl Zeiss) equipped with a Chroma no.
41014 GFP Filter (Chroma Technology Corp.). Photomicrographic recording was performed using a Sony DKC5000 digital photo camera
(Morrel Instruments Inc.) interfaced with Adobe Photoshop v. 4.01 (Adobe Systems, Inc.)
Gene Disruption and Protein A Tagging of MLP1 and MLP2
All yeast strains were derived from W303 (Thomas and Rothstein, 1989;
Table I). Gene replacement of MLP1 and MLP2 was accomplished using
published methods (Rothstein, 1990
; Aitchison et al., 1995a
,b). MLP1 was
replaced with URA3 by generating a PCR product containing the entire
URA3 gene flanked by 75 nucleotides directly upstream and 75 directly
downstream of the MLP1 coding region. A similar procedure was followed to disrupt MLP2 with HIS3. A haploid strain carrying disrupted
copies of both MLP1 and MLP2 (CSDC09
) was constructed by mating
CSDC03
and CSDC05a and subsequent sporulation. Haploid strains of
opposite mating types carrying the double deletion of MLP1 and MLP2
were mated to generate a homozygous diploid strain (CSDC13a/
;
mlp1&mlp2
hd). In all cases correct integration and segregation of each
individual disruptions were verified by PCR analysis of the genomic
DNA. The expression of Mlp1p in wild-type and mutant strains was analyzed by immunoblotting of whole yeast cell lysates and by indirect IF microscopy using mAb148G11.
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For protein A tagging of Mlp1p and Mlp2p, PCR products were generated that contained the coding region for four and a half IgG-binding
repeats of protein A followed by a HIS3, URA3 cassette flanked by 75 nucleotides immediately upstream and 75 nucleotides immediately downstream of the stop codon (i.e., excluding the stop codon itself) of each
gene (Aitchison et al., 1995a,b).
In Vivo Import and Diffusion Assays
The in vivo import assay was performed as described (Shulga et al., 1996).
In brief, wild-type W303 and mlp1&mlp2
hd cells expressing GFP (Cody
et al., 1993
) fused to the SV40 large T antigen NLS were incubated in dextrose-free medium containing 10 mM each of 2-deoxy-D-glucose and sodium azide at 30°C for 45 min. At the end of the incubation, cells were
washed once and incubated in dextrose-containing medium at 30°C to allow reimport of the substrate into the nucleus. The number of normal cells
showing a clear accumulation of GFP-NLS in the nucleus (nuclear cells)
and the number of cells in which the reporter was cytoplasmic were
counted at each time point. At least 40 independent cells were scored per
time point. At least four independent sets of cells were counted to construct the graph presented in Fig. 8. The results are presented as the percentage of cells presenting nuclear signal as a function of time. Linear regression lines were drawn through the linear portion of each curve using
KaleidaGraph and the slope of these straight lines were used to estimate
the relative import rates.
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Passive diffusion assays were performed as follows. pGFP-LEU transformed wild-type and mutant cells were grown as described (Shulga et al.,
1996). Cells were harvested by centrifugation and resuspended in one-fifth
of the initial volume of selective medium. Resuspended cells were held at
4°C until use. The diffusion assay was started by centrifuging the cells,
washing them once in sterile water, resuspending them in 1 vol of 10 mM
sodium azide and 10 mM deoxyglucose in dextrose-free selective medium
and finally placing them at 30°C. Aliquots were taken at each time point
and scored as described above. At least 3 independent sets of 40 cells were
scored at each time point. The relative passive equilibration rates were estimated as described for the import rates.
Overexpression of MLP1
To achieve the overexpression of Mlp1p in yeast cells, the MLP1 gene was
inserted into the pYES2 yeast expression plasmid (Invitrogen Corp.)
downstream of the GAL1/10 inducible promoter. The unique BspHI site
located at nucleotide position 4958 of pYES2 was disrupted using the Klenow fragment of DNA polymerase I to generate pYES2-no BspHI. DNA
from the clone
PM-5620 (Olson et al., 1986
; ATCC catalog number
70598; America Type Culture Collection) containing a yeast genomic fragment of ~16,800 bp from chromosome XI (chromosome bp coordinates:
611843-628638), was used as the template to generate a ~650 bp PCR
product containing the 5' region of MLP1 from nucleotide position
12
(relative to the first bp of the coding region) to nucleotide position +634 flanked by a BamHI site at the 5' end. This PCR product was cut with
BamHI and EcoRI (site located at nucleotide position +572 of the MLP1
coding region) and inserted into the BamHI and EcoRI sites of pYES2-no
BspHI, to generate pYES2-570MLP1. Finally, an ~5,900-bp BspHI fragment of
PM-5620 that contains the entire MLP1 coding region except
the first 8 bp, was inserted into pYES2-570MLP1 that had been linearized
with BspHI (site located at nucleotide position +8 of the MLP1 coding region) to generate pGALMLP1.
pGALMLP1 was transformed into W303 cells by electroporation and transformants were selected taking advantage of the URA3 selectable marker present on the plasmid. Only freshly transformed cells were used for each experiment. To induce the expression of MLP1, W303/ pGALMLP1 cells were grown to mid-logarithmic phase in selective medium containing 2% D(+)-raffinose (referred to as raffinose throughout the text), transferred to selective medium containing 1% raffinose and 2% galactose and incubated at 30°C for up to 4 h. For repression of MLP1 expression, W303/pGALMLP1 cells were grown in raffinose as above, transferred to selective medium containing 1% raffinose and 2% dextrose and incubated for 4 h at 30°C.
Miscellaneous
SDS-PAGE and immunoblotting were performed essentially as described
(Rout and Blobel, 1993). The intensity of bands on immunoblots was
quantified using the ImageQuant v.1.1 software in the PhosphorImager system (Molecular Dynamics), when a Cy5-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories) was used. Occasionally, computer images of enhanced chemiluminescence signals on
photographic films were quantified using the gel plotting macro of NIHImage v.1.60 (Research Services Branch, National Institutes of Health,
Bethesda, MD).
Cells were prepared for indirect IF microscopy using the procedure of
Kilmartin and Adams (1984) with the modifications of Wente et al. (1992)
and Kilmartin et al. (1993)
. Double labeling with mAb148G11 and a polyclonal rabbit anti-Nup159p antibody (Del Priore et al., 1997
) was visualized using Cy3-labeled polyclonal donkey anti-rabbit IgG (cross absorbed
against rabbit IgG) and DTAF-labeled polyclonal donkey anti-mouse
IgG (cross absorbed against mouse IgG; Jackson ImmunoResearch Laboratories). In all single labeling experiments, the bound antibody was visualized using Cy3-labeled polyclonal donkey anti-mouse or anti-rabbit IgG (Jackson ImmunoResearch Laboratories). The staining conditions were as described (Wente et al., 1992
). Photomicrographic recording was
performed using a Zeiss Axioplan2 microscope (Carl Zeiss) equipped
with a Photometrics SenSys A2S digital photo camera (Photometrics) interfaced with IPLab Spectrum p v. 3.1.1c (Signal Analytics).
The growth competition experiment was performed following published procedures (Smith et al., 1996; Rout et al., 1997
; Thatcher et al.,
1998
).
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Results |
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A Screen for Non-NPC Proteins Associated with the NE
Mutations or deletions of the genes that encode for certain
nucleoporins (for example, Nup133p, Nup120p, Nup145p,
and Nup159p) can cause the NPCs to accumulate at one
side of the NE, giving rise to tight clusters that are easily
identified as spots or patches when cells from such strains
are stained with a nucleoporin specific antibody (Doye et
al., 1994; Wente and Blobel, 1994
; Aitchison et al., 1995a
;
Heath et al., 1995
; Kraemer et al., 1995
; Li et al., 1995
;
Pemberton et al., 1995
). It was reasoned that proteins only
partially associated with the NPC, or localized to areas of
the NE that are not in close contact with the NPC, would
either fail to cluster or would only partially cluster with the
NPCs in these strains. Isolated yeast NEs prepared as previously described (Strambio-de-Castillia et al., 1995
) were
used to produce a panel of 114 anti-NE mAbs that were
screened by an assay based on this NPC clustering phenomenon. The indirect IF pattern generated by each of the
individual mAbs on fixed whole wild-type yeast cells was
compared with the staining pattern obtained on a yeast strain carrying a gene disruption in the NUP133 gene
(Pemberton et al., 1995
). The anti-NE antibody, mAb148G11, was identified by this screen as recognizing an antigen that appeared to only partially cluster in the NUP133
disrupted strain. This mAb recognized a protein that runs
as a single band of ~220 kD (apparent molecular mass;
p220) on immunoblots (see Fig. 3).
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Both wild-type yeast and NUP133 deleted cells were
double stained with mAb148G11 and a rabbit polyclonal
antibody against the nucleoporin Nup159p (Fig. 1). In
wild-type cells Nup159p and p220 colocalize to a great extent although this colocalization is not absolute. In particular, some areas of the NE are devoid of one or the other
signals and some cells show some nucleoplasmic staining
with mAb148G11 (Fig. 1, wt, p220 and merge, e.g., the three top cells). This raises the possibility that p220 may
also be found in the nuclear interior. In the clustering
strain the difference in the localization of Nup159p and
p220 is striking (Fig. 1, NUP133). In these cells Nup159p
clearly forms tight clusters localized at the nuclear periphery consistent with its localization at the NPC, while p220
appears to be localized in large patches or even in continuous rims at the nuclear periphery that only partially overlap with the NPC clusters. Furthermore, Nup159p signal
is closer to the cytoplasm than the p220 signal (Fig. 1,
NUP133, merge). This is consistent with the localization of p220 by IEM (see below) and is in accord with results
recently obtained in Xenopus oocytes (Shah et al., 1998
).
In addition, similarly to data obtained with different spindle associated markers (Rout and Kilmartin, 1990
), these
findings demonstrate that it is possible to obtain IF localization to subregions of the NE even in yeast. Similar results were obtained when the localization of Mlp1p and
Nup159p were compared in a NUP120 knock-out strain (data not shown; Aitchison et al., 1995a
).
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Isolation of the Gene Encoding p220
The gene encoding p220, mAb148G11, was identified as
MLP1 (Kolling et al., 1993). MLP1 encodes for a protein
of 1875 AA with a predicted molecular mass of 218 kD
cloned on the basis of its cross-reactivity with a mAb recognizing human platelet myosin (Kolling et al., 1993
). This
nonessential protein was hypothesized to have a nuclear
function on the basis of its subcellular localization (see
Discussion). The major structural features of Mlp1p, the position of the cloned fragment of the gene and of the
epitope of mAb148G11, are shown in Fig. 2 A. The NH2-terminal ~80% of the protein is predicted to have a high
-helical content and contains the heptad-repeats pattern
characteristic of coiled-coil proteins. The COOH-terminal
~400 amino acid residues of the protein are predicted to
form a globular tail rich in phenylalanine and proline residues (Fig. 2 A, P/F rich region). The sequence with the
highest degree of similarity to MLP1 based on FASTA
analysis (28% identical and 66% similar) was the uncharacterized yeast open reading frame (ORF), YIL149c (referred to as MLP2 in Fig. 2 B). YIL149c is expected to encode a protein of 1,680 AA with a predicted molecular
mass of 195 kD. MLP1 and YIL149c belong to a duplicated chromosomal region of the yeast genome (Block 38;
Wolfe and Shields, 1997
) present both on Chromosome XI
and on Chromosome IX. The similarity between Mlp1p and
Yil149p extends over the whole amino acid sequence and
is underscored by the similarity of the overall predicted
secondary structure of the proteins. Yil149p also contains
a phenylalanine and proline rich COOH terminus. Based
on its similarity to Mlp1p and the fact that MLP1 and
YIL149c appear to have arisen from a genome duplication
event, we propose the name MLP2 for YIL149c (see Discussion). The complete yeast genomic database (Clayton
et al., 1997
) contains no other putative homologues of
MLP1.
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Mlp1p and Mlp2p are also similar to the vertebrate and
Drosophila Tpr proteins (Frasch et al., 1988; Mitchell and
Cooper, 1992
; Byrd et al., 1994
; Zimowska et al., 1997
; Fig.
2 B). These proteins are associated with structures found
on the nuclear side of the NPC and may be involved in facilitating nucleocytoplasmic transport (Cordes et al., 1997
;
Zimowska et al., 1997
; Shah et al., 1998
). The main difference between Tpr and Mlp1p is that the COOH-terminal
tail (excluding the predicted coiled-coil region) of Tpr is
relatively much longer, spanning the final 30% of the protein, and has a much more marked acidic character (Byrd
et al., 1994
; Zimowska et al., 1997
). A recently identified S. pombe uncharacterized ORF (SPC162.08c) also shows significant similarities to MLP1, MLP2, and Tpr.
Mlp1p Is Associated with Intranuclear Filaments that Are Localized at the Interface between the NPCs and the Nuclear Interior
Results from the indirect IF analysis shown in Fig. 1 demonstrated that Mlp1p is localized at the NE in areas that
are only partly occupied by NPCs, and that it cannot be
only a NPC constituent, even a peripheral one (e.g., a nuclear basket component). To better understand its localization, the fractionation pattern of Mlp1p was followed
during the preparation of NEs and NPCs (Fig. 3; Rout and
Blobel, 1993; Strambio-de-Castillia et al., 1995
). As expected, the majority of the Mlp1p fractionated with the
nuclei and highly enriched NE fraction, in agreement with
the indirect IF data (Fig. 1). After heparin-extraction of
NEs, nearly all of the NE-associated Mlp1p pool (68% of
the total) was found in the heparin supernatant (Fig. 3, NE
Prep, fractions 13 and 14), demonstrating it is not strongly
associated with the membrane. Only some of Mlp1p cofractionated with the enriched NPC preparation (Fig. 3,
E-NPCs), suggesting a partial or weak association of this
protein with NPCs, again consistent with the indirect IF
results (Fig. 1). It is important to note at this point that
Mlp1p represents the first NE-associated protein to display such a subcellular fractionation pattern (see Discussion).
The ultrastructural localization of Mlp1p was investigated by pre-embedding labeling IEM using mAb148G11
on both isolated NEs (Fig. 4 A, B, and B') and isolated
whole nuclei that had been subjected to mild osmotic
shock to expose the nuclear interior (Fig. 4 C). It should
be remembered that using this procedure only the position
of the epitope of the antigen is being localized rather then
the position of the protein as a whole. On the scale of the
immunostained structures here this may not actually reflect the extent of the localization of the entire protein.
A second important caveat of many immunolocalization
techniques is that the dimensions of the antibody-gold
conjugate have to be taken into account in determining
the precise localization of the epitope. Nonetheless, on
isolated NEs labeled with mAb148G11, gold particles were found almost exclusively on the nuclear side of
the NE often though not always in the vicinities of NPCs.
The average of the distance between gold particles and the
nearest NPC was 66 ± 19 nm on the y axis and 41 ± 34 nm
on the x axis (n = 50; Fig. 5, B and C). On numerous occasions the gold appeared to be associated with fibrillar
structures stretching from the nuclear side of the NE towards the nucleoplasm (Fig. 4 B', arrow). This was in marked contrast to the labeling of Nup159p, a nucleoporin
known to be associated with the cytoplasmic fibrils attached to the outer ring of the NPC (Fig. 5 A). In this case,
and consistent with published results (Kraemer et al.,
1995), the gold particles were found significantly closer to
the NPC (Y = 33 ± 13 nm; X = 8 ± 8 nm; n = 50; Fig. 5
C). The results obtained with the Nup159p control demonstrate that the absence of Mlp1p signal on the cytoplasmic side of the NE can not be due either to a lack of accessibility or to gross alterations or damage of the NE.
|
|
The distribution of the Nup159p control in the isolated
whole nuclei was unchanged with respect to the NEs (Y = 37 ± 11 nm; X = 12 ± 14 nm; n = 14; Fig. 5 C, Nuclei).
While the majority of the Mlp1p signal in whole nuclei was
found in the immediate vicinity of the NE, a significant
fraction was found at a considerable distance into the nuclear interior, sometimes as much as ~300 nm from the
mid-plane of the NE (Fig. 5 C, Nuclei). This resulted in the
average distribution of Mlp1p being further from the mid-plane of the NE than in isolated NEs (Y = 84 ± 56 nm; X = 33 ± 16 nm; n = 50; using the nearest NPC as a referent).
The shorter distance in the case of isolated NEs could be
due to the collapse of filaments during the NE isolation
procedure (see Discussion). Gold was found even further
into the nucleoplasm but it was difficult to establish whether this signal was significantly above background
(Fig. 4 C). No obvious structure was observed in association with gold localized in the interior of the nucleus, although fibrils could again be seen associated with gold
particles found near the NE. Interestingly, in both isolated
NEs and isolated whole nuclei the Mlp1p signal appeared
to extend a maximum of ~120 nm from the cylindrical axis
of the NPCs, which corresponds to the proposed minimum
in vivo inter-NPC distance (Winey et al., 1997). The results of the IEM localization studies are consistent with the indirect IF and immunoblot results presented above (Figs. 1
and 3), and suggest that Mlp1p is associated with filaments
localized at an interface between the nuclear interior and
the NPC.
The COOH Terminus of Mlp1p Is Responsible for Its Nuclear Localization
To determine which region of Mlp1p is responsible for its nuclear localization, the coding sequence of the protein was roughly divided in three thirds (NH2-terminal 1, NT1; NH2-terminal 2, NT2; and COOH-terminal, CT; Fig. 2 A) and each third was GFP-tagged. In addition, a short sequence localized at the non-coiled-coil COOH terminus was selected because of its high lysine and arginine content and hence its similarities to known NLSs, and was also GFP-tagged (putative NLS, pNLS; Fig. 2 A). The intracellular localization of GFP was determined in living cells expressing either GFP alone or the GFP-tagged fragments of Mlp1p (Fig. 6). As expected, untagged GFP had a predominantly cytoplasmic distribution even though it was not excluded from the nucleus. A similar distribution was observed with cells expressing GFP fused to the both of the NH2-terminal thirds of Mlp1p and to the putative NLS. Interestingly however, the COOH terminus of Mlp1p was able to direct the targeting of GFP to the nucleus but not to the NPCs. This suggests that it contains a bona fide NLS that has no obvious homologies to other NLSs, and is not sufficient for NPC association.
|
Mlp2p Resembles Mlp1p in Its Fractionation Behavior and Cellular Localization
To determine the subcellular localization of Mlp2p, the
gene was genomically tagged with an in-frame COOH-terminal fusion of the IgG binding domains of protein A
(pA; Aitchison et al., 1995a). As a control, MLP1 was similarly tagged. Highly enriched NEs fractions were prepared from the Mlp1p- and Mlp2p-tagged strains (Strambio-de-Castillia et al., 1995
) and the fractionation pattern of the proteins was assessed on immunoblots. As expected, the fractionation pattern of Mlp1p-pA was indistinguishable from the one observed in Fig. 3 (data
not shown). Similarly, Mlp2p-pA cofractionated with the
highly enriched NE fraction (Fig. 7 A, fraction 10) but was
almost entirely stripped off by the heparin treatment (Fig. 7 A, fractions 13 and 14). The tagged strains were used to
determine the subcellular localization of the proteins by
indirect IF microscopy (Fig. 7 B). Both proteins were
found localized predominantly at patches found at the nuclear periphery similar to that previously observed for
Mlp1p (Fig. 1). The localization of Mlp1p and Mlp2p in
tagged strains was also determined by preembedding IEM
on isolated NEs. Again these two proteins displayed a very
similar localization pattern to that observed for Mlp1p
(data not shown). These observations are consistent with
the hypothesis that Mlp1p and Mlp2p are functional homologues in accordance with their structural similarities
and their genetic interaction (see below).
|
Double Deletions of MLP1 and MLP2 Cause a Marked Decrease in the Yeast Comparative Fitness
The entire coding regions of both MLP1 and MLP2 were
individually disrupted in the diploid yeast strain W303 by
integrative transformation of the URA3 and HIS3 genes
respectively. Each heterozygous diploid strain (mlp1::
URA3/+ and mlp2::HIS3/+) was sporulated and tetrads
were dissected. In both cases four viable spores from most
tetrads were observed, demonstrating that neither of these genes is essential and confirming and extending published
results (Kolling et al., 1993). Immunostaining with mAb148G11 revealed that the punctate IF staining pattern
and the ~220-kD immunoblot band recognized by both
antibodies was absent in mlp1::URA3 cells but was present in mlp2::HIS3 cells (data not shown). This confirmed that mAb148G11 binds specifically to Mlp1p and demonstrated that mAb148G11 does not cross-react with Mlp2p.
Segregants of opposite mating types carrying the individual disruptions as confirmed by both phenotypic and
genotypic analyses were mated and sporulated. Spores
were isolated that carried both selectable markers demonstrating that the double knock-out of MLP1 and MLP2 is viable.
To assess the degree of selective disadvantage conferred
by individual and double disruptions in the MLP1 and
MLP2 genes, the mlp1, mlp2
, and mlp1&mlp2
strains
were each grown competitively with their wild-type counterpart in rich medium (Smith et al., 1996
; Rout et al.,
1997
; Thatcher et al., 1998
). While mlp1
and mlp2
competed successfully with wild-type, the strain harboring the
double deletion lost ground rapidly even though it was initially added in twofold excess and appeared to be eliminated from the population after 30 generations (data not
shown). These results demonstrated that mlp1&mlp2
had a fitness defect relative to the parental stock equal to
24% (selection coefficient 0.235 ± 0.021; Thatcher et al.,
1998
) and was effectively non-viable outside the protected
laboratory environment. These results also indicated that
MLP1 and MLP2 are homologous.
Deletion of MLP1 and MLP2 Affects the Efficiency of Nuclear Import
To investigate the possibility that Mlp1p and Mlp2p may
be involved in transport of molecules in and out of the nucleus, an in vivo import assay was performed as described
by Shulga et al. (1996). This assay allows the detection of
kinetic defects in the import rates of a NLS-GFP reporter
(Fig. 8 A). In this assay logarithmically growing yeast cells
constitutively expressing NLS-GFP are harvested and poisoned in order to block the production of energy. Under
these conditions the active import of the NLS-GFP reporter into the nucleus is dramatically reduced resulting in the equilibration of the GFP signal between the nucleus
and the cytoplasm by passive diffusion across the NPC.
When the metabolic inhibitors are removed and the cells
are allowed to recover, NLS-GFP is once again rapidly imported in the nucleus. The relative rates of accumulation
of the mutant strain are compared with the ones found
with wild-type, revealing any defect in the mutant's efficiency of nuclear import. The steady state distribution of
NLS-GFP was indistinguishable in homozygous diploid
cells carrying a double deletion of MLP1 and MLP2 as
compared with wild-type (data not shown). Nevertheless,
mlp1&mlp2
hd (homozygous diploid) cells displayed a
markedly slower relative accumulation rate of NLS-GFP into the nucleus with respect to their wild-type counterpart, 13.5 ± 0.2%/min (wild-type) versus 8.9 ± 0.1%/min
(mlp1&mlp2
hd). Significantly, when double mutant cells
were allowed to recover for extended periods of time
(up to double the time required for wild-type), the initial
equilibrium distribution of reporter protein was regained,
again indicating that the efficiency of import and not its
steady state balance was affected by the absence of Mlp1p and Mlp2p. The rates of passive equilibration of the NLS-GFP reporter during the incubation with the metabolic inhibitors were also measured in both wild-type and mutant
cells (Fig. 8 B). In this case, mlp1&mlp2
hd displayed a
significant increase in the relative passive nuclear egress
rates of the NLS-GFP reporter as compared with wild-type,
8.75 ± 1.0%/min (wild-type) versus
13.1 ± 0.6%/ min (mlp1&mlp2
hd). A model that could explain both
the import and the diffusion assay results is presented in
Fig. 8 C. The basic assumption of this model is that the
rates of passive diffusion of the reporter across the NPC
are constant, both in the presence and in the absence of
metabolic inhibitors (i.e., they do not require NTP hydrolysis). Consequently, during recovery, after the removal of
the metabolic inhibitors (
Inhibitor), the rate of active
import becomes greater than the diffusion rate and the net
effect is accumulation of the transport substrate into the nucleus. If the rate of import is lower in mlp1&mlp2
hd
cells with respect to wild-type the prediction is that mutant
cells will take a longer period of time to reach the steady
state levels of nuclear accumulation of the substrate, and
this is exactly what is observed. On the other hand, during
the incubation with the inhibitors (+ Inhibitor), the rate of
active import is drastically reduced in both wild-type and
mutant cells. However, in the mutant cells this residual import is even further compromised as compared with the
wild-type cells. As the diffusion rate remains constant this
will appear as an overall faster egress rate from the nucleus in the mutant cells.
The involvement of Mlp1p and Mlp2p in active nuclear
export was investigated using two steady state assays (data
not shown). In the first assay, the subcellular distribution
of poly(A)+ RNA was analyzed by in situ hybridization
using digoxigenin-labeled oligo(dT)30 as a probe (Wente
and Blobel, 1993). In the second assay, the steady state distribution of a nuclear export sequence (NES)-GFP was
studied by direct fluorescent microscopy (Stade et al.,
1997
). In both cases no effect on export was detected at
steady state and the double mutant cells appeared indistinguishable from wild-type.
Overexpression of Mlp1p
When Mlp1p was expressed from a high copy number
2 µm plasmid, the anti-Mlp1p antibody used by Botstein
and coworkers recognized intensely staining dots and
sometimes rings localized adjacent to the nucleus (Kolling
et al., 1993). This localization does not correspond to the
native localization of Mlp1p discussed in this manuscript
(see above). Mlp1p was overexpressed in W303 cells in order to investigate whether this discrepancy could be accounted for by different levels of expression of this protein. The entire coding region of MLP1 was subcloned in a
2 µm-based yeast expression vector under the control of
the GAL inducible promoter (see Materials and Methods).
Using this plasmid (pGALMLP1), it was possible to overexpress Mlp1p at least ~100-fold over wild-type levels as demonstrated by semi-quantitative immunoblotting performed with mAb148G11 (Fig. 9 A). Strikingly, overexpression of Mlp1p at these levels was not lethal as demonstrated by the ability of cells carrying pGALMLP1 to grow
for days on galactose (data not shown). Cells containing
this construct were either induced with galactose for various periods of time or repressed with dextrose for 4 h and
analyzed by indirect IF microscopy using mAb148G11 to
reveal the localization of Mlp1p (Fig. 9 B). As expected,
cells in which the expression of the exogenous copy of
MLP1 was repressed with dextrose (Fig. 9 B, 4 h [Glu])
showed a staining pattern very similar to the one observed
in wild-type cells. In contrast, induced cells showed an increase of the Mlp1p-specific signal as a function of the induction time. Initially, small dots (1-4 per cell) could be
seen at the nuclear periphery and a clear punctate rim pattern could still be distinguished in most cells (Fig. 9 B, 0 h
[Gal]). Subsequently, these dots appeared to coalesce and
generally gave rise to one prominent circular patch per cell
and the nuclear rim pattern became increasingly more diffuse (Fig. 9 B, 0.5-2 h [Gal]). Finally, the large patch grew
to occupy most of the nuclear interior (Fig. 9 B, 4 h [Gal]).
This staining pattern closely resembled the one described
by Kolling et al. (1993)
, thus reconciling the apparent difference. Careful examination of the overexpressing cells
showed that the intense nuclear DAPI signal was either
much reduced of excluded from the overexpression spots.
It often appeared that the growing Mlp1p spheroids had pushed the chromatin aside, leaving clear indentations in
the chromatin or in some cases even compressing it
between two spheroids (Fig. 9 C). Cells overexpressing
Mlp1p were observed by thin-section transmission EM
to reveal whether any novel structure could be detected
(data not shown). As a comparison, cells that had been grown in dextrose to repress the expression of the non-chromosomal copy Mlp1p were also analyzed using the
same technique. Extensive electron-dense fibrillogranular
networks that extended from different areas of the NE towards the nuclear interior were observed in cells grown in
galactose but were absent in cells grown in dextrose. To
determine if the electron-dense networks present in induced cells were indeed formed of large accumulations of
Mlp1p, isolated nuclei from cells grown in both galactose
(Fig. 10) and dextrose (data not shown) were immunostained with mAb148G11 and prepared for IEM using
the same pre-embedding labeling technique used above. As expected, the dense fibrillogranular networks present in induced cells specifically stained with mAb148G11
demonstrating that they contain large quantities of Mlp1p
and that the Mlp1p spheroids are highly permeable to
large macromolecules such as the antibody-gold conjugate. In cells grown in dextrose, Mlp1p appeared to have a
localization that was indistinguishable from wild-type.
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Discussion |
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The clustering of yeast NPCs within characteristic patches
in the NE induced by the deletion of certain nucleoporins
has previously been used to confirm the association of various proteins with NPCs (Grandi et al., 1995; Aitchison et
al., 1996
; Simos et al., 1996
; Siniossoglou et al., 1996
; Rout
et al., 1997
). Here we used this clustering assay to screen a
bank of monoclonal antibodies raised against NE proteins,
and isolate those which recognize NE components not associated with the NPC from those recognizing NPC components and nuclear transport factors. We expected to find mainly integral nuclear membrane proteins, analogous to
the LAPs or LBR proteins in vertebrate NEs (Senior and
Gerace, 1988
; Worman et al., 1988
, 1990
; Foisner and Gerace, 1993
). Instead, a monoclonal antibody was found
which identified a protein, Mlp1p, associated with the nuclear periphery of the NPC and NE and extending into the
nuclear interior. Sequence comparisons of the predicted
amino acid sequence of Mlp1p with the entire GenBank
database revealed numerous proteins with strong similarities. The most significant was a second yeast ORF predicted to have arisen as a chromosomal duplication event
(Wolfe and Shields, 1997
). We showed this to encode a redundant homologue of Mlp1p which we therefore named Mlp2p. Next, the vertebrate protein Tpr, its probable
Drosophila homologue, and an uncharacterized S. pombe
ORF showed strong similarity to Mlp1p and Mlp2p along
their entire lengths. Though sequence similarity alone is
not necessarily definitive, given the similarities in their localization and possible roles in nuclear transport, we conclude that Mlp1p and Mlp2p are the yeast homologues of
Tpr (see below; Cordes et al., 1997
; Zimowska et al., 1997
;
Bangs et al., 1998
; Cordes et al., 1998
; Shah et al., 1998
).
This represents the first identification and characterization
of Tpr homologues in yeast, setting the stage for studies
that will hopefully elucidate the functions of this protein in
a genetically and molecularly tractable system.
The Tpr homologues are predicted to consist of a coiled-coil NH2 terminus occupying most of the primary sequence. It seems likely that the putative coiled-coil domain
forms an extended structure that may in turn be involved
in organizing higher order homopolymers (for example,
filaments). In contrast, the COOH terminus may interact with heterologous factors and anchor these polymeric structures to the NPCs or to the nuclear interior (see also
below). Despite its name, based on its cross reactivity
with a mAb against myosin (most likely accounted for by
the large coiled-coil NH2-terminal domain; Kolling et al.,
1993), Mlp1p does not belong to the myosin family due to
the different organization of the structural domains along
the primary sequence of the protein.
IF microscopy of the NPC clustering strains shows a partial coclustering of Mlp1p with nucleoporin markers, indicating a significant fraction of Mlp1p is associated with
the NPCs. Our IEM experiments localizing Mlp1p and
Mlp2p to the nuclear face of the nuclear envelope in yeast
agree with the recent results obtained with vertebrate Tpr
(Cordes et al., 1997). Our IEM localization also suggests
that, like Tpr, Mlp1p and Mlp2p form extensive filamentous structures radiating into the interior of the nucleus
from foci at the NPC periphery, perhaps attached at the
distal ends of the NPC baskets or fishtraps (Ris and Malecki, 1993
; Cordes et al., 1997
; Zimowska et al., 1997
).
Also like Tpr, Mlp1p penetrates deep into the yeast nucleus, as far as half of the nuclear radius, and is thus potentially in contact with at least 80% of the chromatin. Interestingly, a significant fraction of Mlp1p failed to cocluster with NPCs, remaining instead as numerous punctate foci
distributed around the nuclear periphery. However, virtually all of Mlp1p cofractionated with the NE fraction, and
the majority of Mlp1p signal was in close proximity to the
NE inner membrane by IEM. Taken together, this suggests that there is also a portion of Mlp1p not associated
with NPCs but distributed around the interporous regions
of the NE inner face. This may differ from Tpr, which was
reported to be absent from these interporous regions, although other studies have found structures believed to be
composed of Tpr interconnecting between NPCs over the
inner NE surface (Ris and Malecki, 1993
; Cordes et al.,
1997
). The punctate IF staining pattern of Mlp1p in regions of NE devoid of nucleoporin signal (and hence
NPCs) may indicate that it can also organize from non- NPC-associated foci distributed around the nuclear face of
the inner nuclear membrane. In an alternative scenario,
structures formed of Mlp1p would absolutely depend on
NPCs for their nucleation at the NE but would be subsequently able to spread all around the nuclear rim in a non-NPC-dependent manner.
It has long been observed that the NPCs are structurally
continuous with the nuclear interior (Monneron and Bernhard, 1969; Franke and Falk, 1971
). Interconnecting open
channels have been observed radiating from the nuclear
interior towards NPCs (Fawcett, 1981
; Berezney et al.,
1995
), and the movement of proteins and RNAs along distinct intranuclear pathways can be studied both in vivo
and in vitro (Lawrence et al., 1989
; Xing et al., 1993
; Zachar et al., 1993
; Misteli et al., 1997
). These and other similar observations have led to speculations that efficient exchange of material between the nuclear periphery and the
nuclear interior could occur along a filamentous network
of tracks. (Blobel, 1985
; Lawrence et al., 1989
, 1993
; Meier
and Blobel, 1992
, 1994
; Xing et al., 1993
; Berezney et al.,
1995
; Ris, 1997
; Pederson, 1998
). Indeed, one model proposes the existence of a nucleoskeleton (defined as a nonchromatin intranuclear structural framework) composed of a filamentous network organized from the nuclear periphery and playing a major role in the direction of nuclear
trafficking to and from the NPCs (Berezney et al., 1995
).
Until recently, the existence of tracks had remained unproven, as no clear candidates for track components had emerged.
A case can be made for the Mlp1p/Tpr family of proteins meeting some of the characteristics expected of nucleoskeletal track components. As one would expect for
track proteins they are localized further away from the
NPCs than the most peripherally described structures of
the NPCs (the nuclear baskets and cytoplasmic filaments) and penetrate most of the volume occupied by chromatin
(as discussed above; Ris and Malecki, 1993; Cordes et al.,
1997
; Ris, 1997
). Their peripheral association with the
NPC is emphasized by the partial fractionation of Mlp1p
with isolated NPCs, though an independent tight association with the nuclear periphery is suggested by its cofractionation with enriched NEs and continuous distribution around the NE even in strains where the NPCs have clustered. Furthermore, such components would not necessarily be stoichiometric with respect to NPC proteins, and not
coassemble with them during NPC assembly. Instead, they
could carry an NLS to be imported separately into the nucleus. Thus, the domain necessary for NPC association
could be separated from that for nuclear localization, as
indeed we showed for Mlp1p and was also recently shown
for vertebrate Tpr (Bangs et al., 1998
; Cordes et al., 1998
).
The Tpr family seems to form filamentous structures of
considerable length (unsurprising considering their predicted coiled-coil structure) that are likely polymeric (Cordes et al., 1997
). Interestingly, the most distal of the intranuclear signal appeared to collapse towards the inner nuclear membrane during the preparation of isolated NEs.
Preliminary data indicate an extreme version of this collapse occurs when NEs are isolated from cells overexpressing Mlp1p (data not shown; see below). Thus it would
seem, as expected of a track component, that interaction
with intact chromatin is required to maintain the normal
distribution of Mlp1p within the nucleus and even opens the possibility of a role for this protein in the maintenance of the nuclear architecture. As lamins are absent from
yeast, it may even substitute in part for their function. The
ability of Mlp1p to self-assemble and form regular polymers that could account for such structural functions remains to be demonstrated and will be the subject of future studies.
As with Tpr, the copious labeling of the COOH-terminal epitope recognized by our antibody indicates that this
part of the protein is free, perhaps to interact with chromatin or transport factors (Cordes et al., 1997). A possible interaction has been found between Tpr and the vertebrate
karyopherin
1 nuclear import factor (Shah et al., 1998
).
Here we show that Mlp1p and Mlp2p are required for the
efficient nuclear import of a substrate of the homologous
yeast karyopherin
1 plus karyopherin
. Although this
represents the first in vivo evidence for a role of this protein family in nuclear transport, it must still be regarded with caution given the potential for pleiotropic effects in
this experiment (see Results). Nonetheless, together such
data support the idea of a direct role for the Tpr family in
nuclear transport by active transport of substrates between binding sites positioned opportunely along the filaments. However, Tpr also coincides with clear channels
extending from the NPC into the nuclear interior (Zimowska et al., 1997
). Overexpression of Mlp1p forms
spheroidal fibrillogranular structures which can occupy
large portions of the nucleus. Overexpression aggregates
such as these usually only display some of the characteristics of the protein under normal conditions, and other
characteristics may be anomalous. However, contrary to
many other proteinaceous aggregates (e.g., Capsey et al.,
1990
) the overexpression spheroids of Mlp1p are surprisingly open to large molecules, as indicated by the apparently unhindered access and accumulation of antibody
conjugated ~10 nm gold (added before fixation, embedding and sectioning for IEM) throughout them. In addition, it is interesting that the Mlp1p spheroids displaced
chromatin as they grew to form large chromatin free regions within the nucleus. One could imagine a polymeric
cylinder of Mlp1p with the properties of these spheroids
projecting from the NPC into the nucleus. Such a structure
would maintain a chromatin free channel that remains
highly permeable to even large proteinaceous particles.
These data are consistent with binding tracks for transport factors, but also raise another possible and less direct role for this protein family; they may maintain efficient nucleocytoplasmic transport by holding open diffusion channels,
for the unhindered intranuclear movement of transport substrates.
It came as a considerable surprise that the deletion of
both yeast Tpr homologues was not lethal. The function of
both proteins must therefore not be essential in yeast, or
other functionally redundant proteins must exist. Either
way, this result holds important consequences for the increasing number of studies underway on the vertebrate
Tpr proteins. Thus despite a reported association with
transport factors (Shah et al., 1998) and a deleterious effect of vertebrate Tpr overexpression on mRNA export
(Bangs et al., 1998
), Tpr homologues are not necessary
to support basic nuclear transport. In vivo experiments
should be conducted to determine if vertebrate cells have
a greater requirement than yeast for Tpr, and what factors
(such as cell size) might contribute to any differences. Further work is also required on the Mlp1p/Mlp2p deficient
strain to find conditions where at least one of these proteins is required for viability. Such experiments may lead to interacting proteins and processes; yeast studies have
been key in showing that many nucleocytoplasmic transport operatives are redundant with numerous subtly overlapping functions, and the Tpr family are proving no exception (Rout et al., 1997
; Wozniak et al., 1998
). The
behavior of these proteins should also be studied in living
yeast cells (such as by using GFP-tagged Mlp1p), taking advantage of available conditional mutant strains, to avoid
some of the problems previously associated with studying potential nucleoskeletal proteins biochemically (Singer
and Green, 1997
; Pederson, 1998
).
In conclusion, we believe that Mlp1p and Mlp2p, together with their multicellular eukaryotic counterparts, satisfy all the criteria expected of non-nucleoporin nucleoskeletal components. We further suggest they represent the strongest candidates to date for the molecular components of the long hypothesized nuclear tracks connecting the NPCs with the nuclear interior. The characterization of the homologues of Tpr in a genetically tractable organism complements the studies of these proteins in vertebrate systems and opens new avenues in the study of nuclear cell biology.
![]() |
Footnotes |
---|
Received for publication 7 December 1998 and in revised form 5 February 1999.
Address correspondence to Dr. G. Blobel, Laboratory of Cell Biology,
Howard Hughes Medical Institute, The Rockefeller University, New
York, New York 10021. Tel.: (212) 327-8096. Fax: (212) 327-7880. E-mail:
blobel{at}rockvax.rockefeller.edu
We are very grateful to J. Aitchison, C. Akey, R. Beckmann, N. Bonifaci,
Y. Chook, E. Coutavas, U. O'Doherty, R. Erdmann, B. Fontoura, J. Helmers, M. Hurwitz, E. Johnson, J. Kilmartin, M. Matunis, L. Pemberton, and S. Smith for many helpful suggestions and discussions throughout
the course of this study. We are deeply indebted to J. Aris, C. Cole, D. Goldfarb, E. Johnson, J. Kilmartin, M. Lewis, L. Pemberton, M. Sogaard,
K. Weis, and R. Wozniak, for providing us with antibodies and other reagents without which this work would not have been possible and to T. De
Lange and J. Karlsreder for assistance in collecting the immunofluorescence data presented here. Many thanks also to R. Beckmann, Y. Chook, and J. Luban for critical reading of the manuscript in whole or in part.
Special thanks go to H. Shio and E. Sphicas for excellent technical assistance in the electron microscopic studies. Our sincerest gratitude goes to
E. Ellison, H. Ijikata, and Y. Oh for invaluable and skillful technical support that was essential for the completion of various parts of the work presented here.
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Abbreviations used in this paper |
---|
GFP, green fluorescent protein; IEM, immunoelectron microscopy; IF, immunofluorescence; MLP, myosin-like protein; NE, nuclear envelope; NES, nuclear export sequence; NPC, nuclear pore complex; NUP, nucleoporin; PVP, polyvinylpyrrolidone.
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References |
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