(Received for publication, July 31, 1995; and in revised form, October 24, 1995)
From the
The 11.5-kDa zinc-binding protein (ZnBP, parathymosin-), a
potent inactivator of 1-phosphofructokinase, is found only in the
cytoplasm of most tissues despite the presence of the putative nuclear
localization signal PKRQKT. Recent reports on nuclear uptake of ZnBP
could not exclude the participation of unspecific diffusion. We show
here that wild-type ZnBP overexpressed in COS cells accumulates
exclusively in the nucleus but that ZnBP with a mutated or deleted
PKRQKT motif appears both in the nucleus and in the cytoplasm. In
contrast, fusion proteins between ZnBP and parts of the endoplasmic
reticulum protein calreticulin required the intact PKRQKT motif for
nuclear import. The motif RKR, located nine amino acids upstream of the
PKRQKT motif, is also involved in the active nuclear import of ZnBP. In
contrast to rat hepatocytes and kidney cells in situ, which
have ZnBP almost exclusively in the cytosol, we find ZnBP in Reuber H35
hepatoma cells and normal rat kidney cells only in the nuclei. Freshly
isolated rat hepatocytes translocate their ZnBP to the nucleus in
<24 h during standard cell culture conditions.
The 11.5-kDa zinc-binding protein (ZnBP) ()was first
described as a factor capable of inactivating 1-phosphofructokinase in
a Zn
-dependent and reversible manner (1) .
ZnBP is found in the cytoplasm of liver, brain, adrenal gland, smooth
muscle, kidney, lung, spleen, and testis, whereas it is only weakly
detectable in skeletal muscle and adipose tissue(2) .
Interestingly, in epithelial cells of the intestinal mucosa and in duct
cells of the exocrine pancreas, it has preferentially been observed in
the nuclei(2) .
As shown by affinity chromatography, ZnBP is
able to bind in a Zn-dependent manner not only to
1-phosphofructokinase but also to other glycolytic and gluconeogenic
enzymes, e.g. aldolase, hexokinase/glucokinase, and others (3) .
The 1-phosphofructokinase-inactivating property has
been located on a tryptic 43-amino acid peptide containing four acidic
clusters of glutamic acid and aspartic acid representing two specific
and two unspecific zinc binding sites(4) . The sequence of this
peptide was used to isolate and sequence both a full-length cDNA clone
from a rat liver library (5) and the corresponding genomic
DNA(6) . The cDNA sequence revealed the identity of ZnBP with
rat parathymosin- (7) and showed the presence of the motif
PKRQKT, which resembles the nuclear localization signal (NLS) TKKQKT of
prothymosin (8) as well as the known ``prototype''
signal PKKKRKV of SV40 large T-antigen(9) .
The injection of
parathymosin- (ZnBP) and prothymosin into Xenopus oocytes
resulted in the accumulation of the injected proteins in the
nucleus(10) . However, it was not excluded in these
experiments, whether the relatively small ZnBP (M
= 11,471) may have entered the nucleus simply by diffusion
through the nuclear pore and thereafter been retained in this
compartment. Therefore, it remained an open question whether the
sequence PKRQKT could function as a NLS and actively translocate ZnBP
into the nucleus. Fusion of human growth hormone lacking the signal
sequence to the N termini of prothymosin and parathymosin-
(ZnBP)
resulted in HeLa S3 cells in a nuclear localization of both fusion
proteins, supporting the idea that prothymosin and ZnBP are imported
into the nucleus in an active manner (11) .
To examine the structure of the putative NLS in ZnBP and its influence on the intracellular localization, we have now overexpressed in COS cells wild-type and point-mutated cDNAs with modified or deleted NLS as well as fusion constructs with a segment of calreticulin (12) lacking its ER import sequence. Since the fusion constructs code for a 48-kDa protein, intracellular distribution of the ZnBP fusion protein should no longer be determined by simple diffusion. Using these constructs, we can show that the import of ZnBP into the nucleus requires an active bipartite nuclear targeting sequence. As ZnBP was found in most rat organs (including liver and kidney) to be restricted to the cytosol(2) , we have also analyzed by immunofluorescence the localization of endogenous ZnBP in NRK and H35 Reuber hepatoma cells and studied the effect of standard cell culture conditions on the distribution of ZnBP in isolated rat hepatocytes. Surprisingly, ZnBP was found almost exclusively in the nucleus not only in the two permanent cell lines, but also in hepatocytes cultured for 12-24 h, indicating a potential role of ZnBP during proliferation or differentiation.
The PCR-generated translated regions of the ZnBP variants contained a 5`-MluI site and a 3`-HindIII site for vector ligation. PCR reactions were performed in a Biomed 60 thermocycler using either 2.5 units of Taq polymerase (calreticulin) or 2.5 units of Pfu polymerase (ZnBPs) and 0.75 µM concentrations of each primer. Primers were annealed at 60 °C for 1.5 min, polymerization time was 1.5 min (1 s extend/cycle) at 72 °C, and denaturation occurred at 92 °C for 2 min. 35 cycles were performed in each reaction. Resulting PCR products of 1076 bp (calreticulin) and 324 bp (ZnBPs) in length were analyzed and purified on 0.7-1% agarose gels. DNAs were digested with SstI/MluI (calreticulin) and MluI/HindIII (ZnBPs).
ZnBP fragments were
subcloned into the SstI/HindIII precut pGEM-4Z vector
(Promega, Heidelberg, Germany). Recombinant colonies were identified by
blue/white color phenotype, and plasmid DNA was restriction analyzed.
Plasmid DNA of pGEM-4Z/ZnBP was then linearized with SstI and MluI, and calreticulin PCR fragments were subcloned into the
vector-ZnBP constructs, resulting in the complete fusion genes. Fusion
gene clones were analyzed by Southern hybridization of various
restriction fragments, and EcoRI/HindIII fragments
were subcloned into pCMV2. To generate the fusion construct
CR-ZnBP-1, the ZnBP fragment of CR-ZnBP-WT was replaced via MluI/HindIII by the two-step PCR product,
ZnBP-
1, mentioned above.
Construction of the dihydrofolate
reductase fusion genes was based on three different dihydrofolate
reductase PCR products, which all coded for the same part of
dihydrofolate reductase (amino acids 2-186). The first contained
an EcoRI site at its 5`-end followed by a Kozak
sequence(15) , an ATG, and restriction sites HindIII
and MluI for later introduction of ZnBP variants. Its 3`-end
contained the motif PKRQKT, a c-myc tag, the stop codon, and a XbaI site. The second PCR product contained the same
5`-structure as the first one, but it possessed only a BglII
site (and no stop codon) at its 3`-end used for fusion with the third
PCR product, which contained a 5`-BglII site and shared its
3`-end with the first PCR fragment. This first dihydrofolate reductase
PCR product was cloned into pCMV2 directly, resulting in vector pD. The
other two were subsequently cloned into pGEM4Z and thereby fused by the BglII sites. The resulting EcoRI/XbaI
fragment (containing a dimeric dihydrofolate reductase fragment) was
subcloned into pCMV2, resulting in vector pDD. The HindIII/MluI sites present in the 5`-region of the
dihydrofolate reductase inserts in pD and pDD were used to introduce
PCR-generated fragments of ZnBP spanning an N-terminal portion (amino
acids 1-34, ZnBP-N) or the central acidic cluster (amino acids
35-72, ZnBP-C). In additional constructs, the entire coding
region of ZnBP in its variants ZnBP-WT and ZnBP-Thr was
subcloned into pD and pDD.
All overexpression constructs were sequenced to verify the fidelity of PCR reactions and fusion borders as well; pCMV2 constructs were used for overexpression studies.
Perfusion
of rat livers was performed as described by Kleinecke et
al.(16) . Fresh hepatocytes were cultivated on
collagen-plated coverslips and maintained in medium M199 (Life
Technologies, Inc.) containing 1.5% (w/v) of both penicillin and
streptomycin, 10M dexamethasone, and
10
M porcine insulin. Cells were grown at
37 °C for 45 min, 4 h, or 24 h. Subsequent immunofluorescence
studies of all cell lines were performed as described below.
For
Northern blot studies, the 936-bp ZnBP cDNA (5) was labeled
with P using the random priming method(17) . Total
RNA from fresh hepatocytes as well as from hepatocytes cultivated for 4
and 24 h was isolated as described in (18) . Northern blotting
onto GeneScreen Plus membranes (Dupont NEN) and hybridization were
performed at 42 °C as described in (14) . Hybridized
membranes were washed twice in 2
SSC (5 min), twice in 2
SSC, 1% SDS at 60 °C (30 min), and twice in 0.1
SSC
at 20 °C (30 min) and exposed to x-ray film for 3-24 h. For
Western blotting, transfected cells were analyzed using purified
anti-ZnBP. Immunoblots of ZnBP-transfected cells, as well as of rat
hepatocyte cell lysates were performed and stained as given in (2) , fusion protein blotting followed standard
procedures(14) , with additional renaturation at 42 °C in
Tris-buffered saline for at least 6 h(2) .
COS, NRK, and H35 Reuber
hepatoma cells as well as rat hepatocytes were fixed in 3%
paraformaldehyde and permeabilized with 0.3% Triton X-100. Double
immunofluorescence was performed using affinity-purified rabbit
anti-ZnBP (diluted 1:50 in phosphate-buffered saline, 10% FCS) or
monoclonal mouse anti-SV40 large T-antibody (Dianova, 1:100 in
phosphate-buffered saline, 10% FCS) following standard procedures. Goat
anti-rabbit fluorescein isothiocyanate conjugate and goat anti-mouse
tetramethylrhodamine isothiocyanate conjugate secondary antibodies
(Jackson ImmunoResearch/Dianova, Hamburg, Germany) were used in 1:200
dilutions in phosphate-buffered saline, 10% FCS. Samples were embedded
in Moviol and visualized at emission wavelengths of 519 nm (fluorescein
isothiocyanate) and 572 nm (tetramethylrhodamine isothiocyanate),
respectively. To visualize ZnBP from rat hepatocytes with
peroxidase-coupled goat anti-rabbit antibodies, endogenous peroxidase
of permeabilized hepatocytes was inhibited by incubation of cells with
0.5% HO
in Tris-buffered saline for 15 min at
room temperature. Incubation with the anti-ZnBP antibody was performed
as described above followed by incubation with goat anti-rabbit
peroxidase conjugate (diluted 1:2000 in Tris-buffered saline, 10% FCS).
The staining reaction was performed with 5 ml of 100 mM citrate (pH 6), 100 µl of N,N-diethyl-p-phenylenediamine (Eastman
Kodak Co.) solution (12 mg/ml in acetonitrile/H
O (4:1)),
and 500 µl 0.3% (w/v) 4-chloro-1-naphtol (in CH
OH and 2
µl 30% H
O
) for 30 min at room temperature.
The reaction was stopped by washing in Tris-buffered saline.
Figure 1:
Mutations introduced into ZnBP. The
C-terminal 12 amino acids of ZnBP and the C-terminal 26 amino acids of
the ZnBP sequence in CR-ZnBP-1 as well as the corresponding cDNAs
are shown. The bipartite nuclear localization signal (see below) is
printed in italics. The asterisks (***) mark the stop
codons. Point mutations on the cDNA and mutated amino acids are printed
in boldface.
By Western
blotting, it was assured that the affinity-purified anti-ZnBP antibody
reacted only with the overexpressed wild-type or mutated ZnBPs (Fig. 2, upper panel), but not with endogenous COS cell
proteins (Fig. 2, lower panel, lane E). Using
this antibody, ZnBP-WT transiently overexpressed in COS cells was found
almost exclusively in the nucleus (Fig. 3A). In contrast,
protein expressed from ZnBP-Stop was observed not
only in the nucleus but also in the cytoplasm (Fig. 3C). Expression products from ZnBP-Ser
and ZnBP-Thr
revealed the same intracellular
distribution as ZnBP-Stop
, i.e. they gave a
prominent nuclear staining with additional fluorescence from the
cytoplasm (Fig. 3, E and G). A significant
difference between the mutants could not be observed. Intracellular
localization of SV40 large T-antigen in the same cells was used as
control for intact nuclei. These results indicate that the putative NLS
(PKRQKT) had influenced the nuclear localization of ZnBP in COS cells,
as mutated and deleted forms of this motif were accompanied by an
additional cytoplasmic localization.
Figure 2:
Western blot of wild-type ZnBP and ZnBP
mutants (upper panel) and of fusion proteins consisting of
parts of calreticulin and wild-type ZnBP or mutated ZnBP (lower
panel) in COS cells. Upper panel, lane A, purified ZnBP
(0.5 µg); lane B, expressed ZnBP-WT; lane C,
expressed ZnBP-Ser; lane D, expressed
ZnBPThr
; lane E, ZnBP-Stop
. Lower panel, lane M, M
markers; lane
A, expressed CR-ZnBP-WT; lane B,
CR-ZnBP-Stop
; lane C, CR-ZnBP-Thr
; lane D, CR-ZnBP-Ser
; lane E, lysate from
nontransfected cells. Cells were homogenized as given under
``Experimental Procedures,'' and 50 µg of total protein
was separated by SDS-PAGE and immunoblotted. Note that ZnBP displays an
apparent molecular mass of 19 kDa in SDS gels(1) . Although
CR-ZnBP-WT is an expression product from clonal DNA, it appears
occasionally as a double band in these blots (lane
A).
Figure 3:
Immunofluorescence of COS cells
overexpressing constructs ZnBP-WT (A and B),
ZnBP-Stop (C and D), ZnBP-Ser
(E and F), and ZnBP-Thr
(G and H). Double-immunofluorescence was performed using
anti ZnBP antiserum (panels A, C, E, and G) and anti-SV40 large T-antigen antiserum (panels B, D, F, and H). All COS cells display SV40
large T-antigen immunoreactivity in their nuclei. Wild-type ZnBP (A) appears almost exclusively in the nuclei, whereas mutant
variants (C, E, and G) are detected also in
the cytosol.
Figure 4: Structure and size of calreticulin-ZnBP fusion genes overexpressed in COS cells. Fusion genes with the location of their functional parts are shown in shaded boxes. Functional protein motifs are printed in italics. ZnBP parts of the constructs are printed in boldface italics. The ATG marks the start and an asterisk marks the stop codon of each open reading frame. The calreticulin lacks the signal sequence as well as the C-terminal acidic clusters and the KDEL motif.
Figure 5:
Immunofluorescence of COS cells
overexpressing constructs CR-ZnBP-WT (A and B),
CR-ZnBP-Stop (C and D),
CR-ZnBP-Ser
(E and F), and
CR-ZnBP-Thr
(G and H). Experiments were
performed as described in the legend of Fig. 3. Panels
A, C, E, and G show results with anti
ZnBP antiserum; panels B, D, F, and H show those with anti-SV40 large T-antigen antiserum. Wild-type
fusion products (A) are located in the nuclei, whereas mutant
fusion variants (C, E, and G) are restricted
to the cytosol, indicating the active role of a NLS of
ZnBP.
Figure 6: Structure and size of dihydrofolate reductase-ZnBP fusion genes overexpressed in COS cells. Fusion genes with the location of their functional parts are shown in boxes. Functional protein motifs are printed in italics. ZnBP-parts of the constructs are printed in boldface italics. The localization of the motif PKRQKT is indicated directly. The ATG marks the start and an asterisk marks the stop codon of each open reading frame.
Figure 7:
Immunofluorescence of COS cells
overexpressing constructs pD (A), pD-ZnBP-WT (B),
pD-ZnBP-Thr (C), pD-ZnBP-N (D), and
pD-ZnBP-C (E). In contrast to experiments described in Fig. 3and Fig. 5, expressed proteins were detected using
the c-myc antibody. Whereas protein expressed from construct
pD, which lacks any ZnBP sequences, was distributed almost evenly
throughout the entire cell (A), addition of parts of ZnBP
resulted in a shift in the localization toward the nucleus (B-E). pD-ZnBP-WT appeared almost exclusively in the
nucleus (B).
Figure 8:
Immunofluorescence of COS cells expressing
constructs pDD-ZnBP-WT (A), pDD-ZnBP-Thr (B), pDD-ZnBP-N (C), pDD-ZnBP-C (D),
and CR-ZnBP-
1 (E). Expressed proteins were detected with
the c-myc antibody. All constructs expressed from pDD (except
for pDD-ZnBP-WT, panel A) were excluded from the nucleus,
thereby demonstrating that the motif PKRQKT is not sufficient to direct
them to the nucleus. Construct CR-ZnBP-
1 (E) demonstrates
the bipartite nature of the ZnBP NLS. Proteins expressed from this
construct remained in the cytoplasm, indicating the necessity of the
short basic cluster RKR nine amino acids upstream the PKRQKT
motif.
Surprisingly, all proteins
expressed from pDD vector (except for pDD-ZnBP-WT) remained in the
cytoplasm, despite their C-terminal motif PKRQKT (Fig. 8, B-D). In contrast pDD-ZnBP-WT, expressing wild-type ZnBP
in the dihydrofolate reductase environment was found almost exclusively
in the nucleus (Fig. 8A). pDD-ZnBP-Thr, as
well as the calreticulin fusion protein of this variant
(CR-ZnBP-Thr
, Fig. 5G), remained in the
cytoplasm (Fig. 8B). This demonstrates that the motif
PKRQKT alone is necessary but not sufficient to function as an active
NLS.
The overexpression of the pD constructs gave a clear hint for a
nuclear retention of ZnBP (Fig. 7). Protein expressed from pD
alone (without ZnBP-sequences but containing the PKRQKT-motif) appeared
equally distributed between the cytoplasm and the nucleus (Fig. 7A). Insertion of wild-type ZnBP (pD-ZnBP-WT)
resulted in a nearly exclusive nuclear localization due to its active
import (Fig. 7B). In contrast to the distribution of
products expressed from pD without ZnBP-insert, all other ZnBP inserts
(pD-ZnBP-Thr, pD-ZnBP-N, and pD-ZnBP-C) led to a clear
shift of the expressed proteins toward the nucleus (Fig. 7, C-E), although the products of the corresponding pDD
constructs had remained in the cytoplasm, indicating the absence of
NLS-mediated transport. Therefore, the nuclear import of pD chimeras
must have resulted from passive diffusion into the nucleus. The
increased nuclear retention of the products of
pD-ZnBP-Thr
, pD-ZnBP-N, and pD-ZnBP-C as compared with
that of pD (Fig. 7A) points to an interaction of
ZnBP-sequences with nuclear components.
Figure 9: Localization of endogenous ZnBP in NRK cells (panel 1), H35 Reuber hepatoma cells (2), and in primary cultured hepatocytes after 45 min (3 and 5), 8 h (4), and 24 h (6) of cultivation. Fresh hepatocytes (45 min, panels 3 and 5) gave only weak nuclear signals, whereas ZnBP appears in the nuclei after 8 h (panel 4) or 24 h (panel 6) of cultivation. In the permanent cell lines (panels 1 and 2), ZnBP is located exclusively in the nuclei. Note, that in the case of hepatocytes the whole cells are seen, whereas in the case of the NRK cells (panel 1) and the H35 Reuber hepatoma cells (panel 2) strong fluorescence from the nuclei is visible, with only weak signals from the cytosol. The dark areas in the nuclei of hepatoma cells represent nucleoli. In panels 1-4, ZnBP was visualized by immunofluorescence, in panels 5 and 6 by staining with N,N-diethyl-p-phenylenediamine. In freshly seeded hepatocytes (panel 5), a moderate staining of the cytoplasm was visible, which, however, could hardly be reproduced using black and white photography.
Figure 10: Levels of ZnBP (panel 1) and transcripts of ZnBP (panel 2) in cultivated rat hepatocytes. Panel 1, Western blots of ZnBP in rat hepatocytes. Lane A, 100 ng of purified ZnBP; lane B, ZnBP in fresh rat hepatocytes; lane C, ZnBP in rat hepatocytes cultivated for 4 h; lane D, ZnBP in rat hepatocytes cultivated for 24 h. Proteins from of total rat hepatocyte lysate (85 µg) were separated and immunoblotted. Panel 2, Northern blots of ZnBP transcripts in rat hepatocytes detected with the 936-bp ZnBP-cDNA. Lane A, fresh rat hepatocytes; lane B, rat hepatocytes cultivated for 4 h; lane C, rat hepatocytes cultivated for 24 h. Total RNA from rat hepatocytes (20 µg/lane) was separated on agarose gels and blotted onto a GeneScreen Plus filter, and ZnBP transcipts were detected by the radiolabeled ZnBP cDNA(5) .
Altogether, our results indicate that proliferating cells like the NRK or H35 Reuber hepatoma cells contain most of their ZnBP within the nucleus, whereas a nonproliferating cell like the freshly seeded hepatocyte retains its ZnBP mainly in the cytosol. Only after several hours of cultivation under conditions where the hepatocytes tend to dedifferentiate(20) , ZnBP undergoes net translocation to the nucleus.
Although previous studies from this laboratory have revealed that ZnBP is located mainly in the cytosol in most tissues(2) , we show here that ZnBP contains a functionally active bipartite NLS. Previous experiments, in which ZnBP had been injected into Xenopus oocytes, had shown the uptake of ZnBP into the nucleus(10) . Clinton et al.(11) demonstrated in HeLa S3 cells a nuclear accumulation of ZnBP and prothymosin fused to a truncated form of human growth hormone. These fusion proteins had molecular masses of about 34 kDa. However, the authors did not analyze whether the proposed nuclear targeting sequence PKRQKT was active, although the fusion protein theoretically was able to enter the nucleus also by diffusion. Indeed, we have shown here that not only ZnBP with a mutated PKRQKT motif but also fusion proteins containing ZnBP linked to dihydrofolate reductase, but lacking an intact NLS, diffuse into the nucleus inspite of their molecular mass of about 34 kDa. Only by constructing even larger fusion proteins with intact or mutated NLS, we could unequivocally show that the NLS is operative. Our results clearly show that ZnBP represents a small protein, capable of diffusion into the nucleus, but possessing an operative NLS. This is different from histone H1. Histone H1 can potentially diffuse into the nucleus and requires an active uptake mechanism sensitive to chilling and energy depletion(21) , but for histone H1 an NLS has not been identified up to now. The participation of a yet unknown protein with an NLS carrying histone H1 by ``piggy-back'' transport cannot be excluded.
While the dihydrofolate reductase expressed from pD is largely found in the cytoplasm and only to a limited extent in the nucleus, the pD expression products containing additional sequences from ZnBP accumulate much more in the nucleus in spite of the fact that none of these constructs have an active (bipartite) NLS. This indicates, that the nuclear/cytoplasmic distribution of ZnBP may be determined not only by passive diffusion or NLS-dependent import but also by nuclear retention, probably resulting from interaction of ZnBP or fragments of ZnBP with nuclear components.
Our results show further that the motif RKR situated nine amino acids upstream from the PKRQKT motif is also involved in nuclear targeting of rat ZnBP. The exchange of the central lysine against an alanine abolished the nuclear uptake of the ZnBP-calreticulin fusion protein in spite of an intact PKRQKT motif. In human ZnBP, one finds the motif LKR instead of RKR(22) , which could indicate that either the KR motif or even the lysine alone is sufficient. Bovine ZnBP, on the other hand, contains the sequence LVR(23) . If this motif were operative as part of a bipartite NLS, it would be in conflict with our observation that the lysine is required for nuclear import. As a cDNA-derived sequence of bovine ZnBP has not been published up to now, a sequencing error cannot be completely excluded. In any case, the sequences in ZnBP from rat and human would be in line with the ``consensus'' sequence for a bipartite NLS(24) , where the upstream situated part of the NLS is composed of 2-3 basic amino acids (e.g. KR in Xenopus nucleoplasmin or KRK in poly(ADP-ribose)polymerase). The motif TKKQKT interpreted as the NLS of prothymosin (8) may also be only part of a bipartite NLS, as 11 amino acids upstream of this motif a KR-motif exists in rat as well as in human prothymosin.
ZnBP-calreticulin fusion proteins contained still the calreticulin sequence PPKKIKDPD. This sequence had been interpreted as a potential NLS by Michalak et al.(12) . However, fusion proteins containing this sequence did not enter the nucleus unless their ZnBP portion contained the intact NLS of ZnBP. This finding could be of importance considering the recent reports of a transcriptional control exerted by intranuclear calreticulin(25, 26) .
We have shown here clearly
that the NLS of ZnBP is operative. Why then does ZnBP exist in most
tissues mainly in the cytosol? One explanation could be that in
differentiated cells, ZnBP normally is bound to other cytoplasmic
proteins, which would block the interaction of the nuclear targeting
signal of ZnBP with importins(27) . The binding of ZnBP to this
blocking protein would have to be overcome to allow for the formation
of free ZnBP, which then could interact with importins and enter the
nucleus. An interaction with such blocking proteins might be abolished
by decreasing the affinity of these proteins for ZnBP, e.g. by
changing its state of phosphorylation or altering the cytoplasmic
milieu or by its proteolytic degradation. Such a mechanism would
resemble the inhibition of nuclear transport of NFB by
IF
B(28) . If this were so, proliferating or
dedifferentiated cells should express a decreased concentration of
binding protein(s) or show conditions of decreased binding affinities
of such binding protein(s) for ZnBP. The fact that ZnBP has been shown
to interact not only with 1-phosphofructokinase but also with several
other cytoplasmic enzymes (3) would support the idea that under
physiological conditions ZnBP can interact also with other cytoplasmic
proteins. This property of ZnBP also makes it necessary to examine the
possibility that ZnBP transports proteins lacking an NLS into the
nucleus by piggy-back transport.
That the cytoplasmic/nuclear distribution of ZnBP undergoes regulation under in vivo conditions is underlined by our following observations. (a) Rat kidney derived NRK cells and rat hepatocyte-derived Reuber H 35 hepatoma cells contain their ZnBP almost exclusively in the nucleus, whereas ZnBP in the corresponding differentiated cells exists in the cytoplasm ( (2) and see Fig. 9). (b) As in intact liver(2) , freshly seeded isolated rat hepatocytes had almost no ZnBP in their nuclei as seen in immunofluorescence experiments. But after 8-24 h of cultivation under standard conditions, almost all ZnBP appeared in the nucleus. These results complete our earlier observation(2) , that in contrast to most other tissues, intestinal mucosa cells in the zona germinativa of the crypts of Lieberkühn contained ZnBP mainly in the nuclei.
Under standard culturing conditions, hepatocytes start relatively soon to dedifferentiate(20) . Furthermore, it has been reported recently, that in human mammary tumor tissue, the levels of prothymosin as well as those of ZnBP had increased dramatically(29) . In view of these observations, we have now begun to examine the question whether and how ZnBP might be involved in processes of cell differentiation and/or proliferation and to identify factors responsible for retention of ZnBP in the cytoplasm of differentiated, nonproliferating cells.