(Received for publication, October 20, 1995; and in revised form, January 3, 1996)
From the
The fungal metabolite brefeldin A disrupts protein secretion and causes the redistribution of the Golgi complex to the endoplasmic reticulum. Previously we isolated six genes that, when present in multiple copies, confer brefeldin A resistance to wild type Schizosaccharomyces pombe. Here we describe the characterization of one of these genes, hba1. This gene encodes an essential protein that shares homology with the mammalian protein RanBP1 and the protein encoded by the Saccharomyces cerevisiae gene YRB1 and contains a peptide motif present in several proteins found within the nuclear pore complex. The protein encoded by hba1 is localized to the nucleus, and it was determined that this protein is phosphorylated in vivo. The characterization of hba1 thus demonstrates a novel mechanism of drug resistance in S. pombe.
A number of pharmacological agents have been used as probes to
examine the processes and mechanisms responsible for intracellular
protein transport and secretion. One of these compounds, brefeldin A
(BFA), ()has been used extensively to investigate the
underlying mechanisms of both protein transport and also maintenance of
intracellular organelles. Addition of BFA to cultured cells results in
the rapid inhibition of protein secretion and the redistribution of
Golgi proteins and membranes into the endoplasmic reticulum
(Lippincott-Schwartz et al., 1989; Doms et al., 1989;
Fujiwara et al., 1988). BFA is thought to exert its effects on
protein transport and Golgi structure by inhibiting the GDP to GTP
exchange on ADP-ribosylation factor (ARF) found on Golgi membranes
(Donaldson et al., 1992; Helms and Rothman, 1992). However,
more recent data using partially purified ARF guanine nucleotide
exchange factor demonstrated that BFA did not inhibit the GDP-GTP
exchange reaction (Tsai et al., 1994). Therefore, it is
unclear whether the nucleotide exchange on ARF is effected by BFA
directly.
We have utilized the fission yeast Schizosaccharomyces pombe in order to identify the target molecule of BFA as well as other proteins capable of conferring BFA resistance. Our previous work has demonstrated that wild type S. pombe is sensitive to the effects of BFA in an analogous manner to that seen in mammalian cells, i.e. inhibition of protein secretion and the disassembly and redistribution of the Golgi complex (Turi et al., 1994). Mutant S. pombe strains that are resistant to BFA were isolated, characterized and found to contain a mutation in either crm1, a gene required for maintaining chromosomal structure, or a second locus termed bar2. In addition, six genes that confer resistance to BFA when present in high copy were also identified (Turi et al., 1994). One of these six genes encoded a homologue of the mammalian transcription factor AP1 termed pap1, while yet another of the six genes encoded a novel multidrug resistance transporter (Turi and Rose, 1995). Here we describe a third gene conferring BFA resistance. The protein encoded by this gene is an essential nuclear phosphoprotein that contains a conserved sequence motif present in proteins that interact with a nuclear GTP-binding protein known as Ran in mammalian cells, or Spi1 and GSP1 in fission and budding yeast, respectively.
The hba1 gene on plasmid pBAR2-1 was localized by subcloning restriction fragments corresponding to the insert into plasmid pFL20 (Losson and Lacroute, 1983). Recombinant plasmids were used to transform wild type S. pombe and BFA resistance examined using a growth or a secretion assay previously described (Turi et al., 1994). Plasmid pBAR2-1H contained a 4.2-kb HindIII fragment that conferred BFA resistance in both assays. To further map the hba1 gene, a 2.7-kb HindIII-EcoRI subfragment of the pBAR2-1 insert was cloned into pFL20 and assayed for BFA resistance.
The wild type hba1 gene was also cloned into pREP4 using the 5` PCR product described above digested with SmaI and PstI and mixed with a PstI to EcoRI (made blunt with Klenow fragment of DNA polymerase) restriction fragment of pBAR2-1H containing the 3` half of the hba1 gene. These two fragments were then ligated into the MscI site of pREP4.
For immunoprecipitation, the labeled cells were disrupted with glass beads in detergent lysis buffer (Rose and Bergmann, 1983) and immunoprecipitates produced using 100 µl of conditioned medium from 9E10 hybridoma (Evans et al., 1985) followed by 1 µl of a rabbit anti-mouse polyclonal antiserum and fixed Staphylococcus aureus. The immune complexes were washed five times with RIPA (Rose and Bergmann, 1983), one time with 50 mM Tris-HCl, pH 8.0, and one time with Tris containing 100 µg/ml RNase. The samples were subjected to electrophoresis on a 10% polyacrylamide-SDS gel. The gel was soaked in water containing 10 mM ATP for 10 min, dried, and exposed to film.
Figure 1: Restriction mapping of pBAR2-1. Restriction map and subclones from pBAR2-1. The locations of restriction endonuclease sites on the original pBAR2-1 plasmid are indicated as vertical bars (abbreviations are: B, BglII; E, EcoRI; H, HindIII). Each restriction fragment was subcloned into pFL20. The resulting plasmid was used to transform the wild type S. pombe strain FWP1. The ability of each plasmid to confer BFA resistance is shown to the right of each fragment.
The DNA sequence of the 4.2-kb fragment was determined. Two long open reading frames were present within this fragment (diagrammed in Fig. 1). The upstream ORF was complete, while the downstream ORF was truncated at the distal HindIII site. The remaining portion of this second ORF was determined by sequencing directly from plasmid pBAR2-1. To determine which of the two ORFs was capable of conferring the BFA resistance, each gene was individually subcloned into pFL20, transformed into wild type S. pombe, and transformants analyzed as before. Only the complete upstream ORF present on the original 4.2-kb HindIII fragment conferred BFA resistance (data not shown). The nucleotide and and amino acid sequence of this ORF, which we refer to as hba1 for hyperresistance to brefeldin A, is shown in Fig. 2. The hba1 coding sequence begins at nucleotide 612 and terminates at nucleotide 1809. The second ORF, which is referred to as dhb1 for downstream of hba1, begins 1268 nucleotides downstream of the hba1 ORF (sequence not shown, but is included in GenBank accession no. U38783). Comparison of the dhb1 ORF to the GenBank data base failed to detect any significant sequence similarity to any other known protein.
Figure 2: Nucleotide and predicted protein sequence of the hba1 gene. The sequence of the entire pBAR2-1H insert was deduced. Shown is the sequence corresponding to the hba1 gene. The putative nuclear localization sequence is underlined. The complete nucleotide sequences of both hba1 and dhb1 have been deposited in the GenBank/EMBL data base under accession number U38783.
The library used to isolate hba1 on pBAR2-1 was constructed from a S. pombe strain possessing a dominant mutation within the bar2 gene, which itself confers BFA resistance. It was possible that the hba1 allele isolated may have contained a mutation. We therefore determined the sequence of hba1 from plasmid pBAR1-2. This plasmid was isolated from a library constructed from a strain harboring a bar1/crm1 mutation in a wild type background, and it would be expected that the hba1 allele isolated from this strain be wild type. Comparison of the hba1 sequence from pBAR1-2 and pBAR2-1 determined that the two sequences were identical, demonstrating that the gene isolated from pBAR2-1 was wild type.
Figure 3: Comparison of amino acid sequence of hba1, CST20, and RanBP1. Amino acids that are identical between all three proteins and denoted by the solid boxes. Hatched boxes denote amino acid residues in which a conservative replacement has been substituted in one of the proteins. The putative nuclear localization domain of Hba1 is located between residues 191 and 214. The sequence data used were taken from Coutavas et al. (1993; accession number L25255) and Ouspenski et al. (1995; accession number X65925).
Several
proteins in addition to RanBP1 and YRB1 were also found to have
significant similarity to the COOH-terminal half of Hba1p. These
proteins included the S. cerevisiae genes NUP2 and an
ORF present on Chromosome IX with sequence similarity to NUP2
(NUP2-like), a Caenorhabditis elegans ORF (Fig. 4) and
three human proteins, two of which were identified by screening a
hippocampal expression library with [P]GTP-Ran
(Beddow et al., 1995) and the other by hybridization with
murine RanBP1 (Bischoff et al., 1995). The latter of the three
human RanBP homologues, subsequently identified and renamed RanBP2
(Yokayama et al., 1995), contains two homology domains and is
similar in structure to the C. elegans ORF. The region of
similarity between all these proteins is centered around two potential
leucine zippers that have been predicted in RanBP1 (Coutavas et
al., 1993).
Figure 4:
A conserved motif found in Ran-binding
proteins is present in Hba1p. The region of sequence similarity between
Hba1p and Ran-binding proteins is shown. The protein sequences were
aligned with the Megaalign program of DNAstar. Sequences used were S. pombe hba1, S. cerevisiae proteins encoded by the CTS20, NUP2, and NUP2-like genes, the mouse
RanBP-1 (L25255), C. elegans open reading frame F59A2 (which
contains both a NH-terminal and COOH-terminal motif), the
human gene RanBPX (which also contains two motifs: a and b) (X83617),
and the human AB1 (U19240) and AB2 (U19248) genes. Closed circles denote amino acid residues that are identical in all the aligned
proteins; open circles denote residues that are highly
conserved between the aligned proteins.
Figure 5: Localization of Hba1p in S. pombe. Indirect immunofluorescence was performed on wild type S. pombe transformed with vector control (pREP4) or with a vector containing the hba1 gene (pREP4-HBA1) or the hba1 gene with the human c-Myc epitope appended to the carboxyl terminus (pREP4-HBA1-Myc) under the control the nmt1 promoter. Panels on the left correspond to 4`,6-diamidino-2-phenylindole, dihydrochloride-stained nuclei. Panels on the right correspond to fluorescence observed when the cells are stained with the anti-c-Myc monoclonal antibody 9E10 and a fluorescein isothiocyanate-labeled secondary antibody. Immunofluorescence was performed as described by Hagan and Hyams(1988), except that spheroplasting was terminated when approximately 50% of the cells became refractory.
To exclude mislocalization by the Myc epitope, we appended the Myc epitope onto the carboxyl terminus of the cytoplasmic protein Obr1 and expressed this construct using the nmt1 promoter (Toda et al. 1992; Turi et al., 1994). The Obr1-Myc fusion was localized throughout the cytoplasm with no evidence of nuclear staining (data not shown). These results demonstrate that the nuclear localization of Hba1p is not due to the presence of the Myc tag. Examination of the deduced Hba1p sequence identified a possible bipartite nuclear localization signal located between residues 191 and 214, which contains the sequence KKFAAGTAVETESGSGKEKENDKK ( Fig. 2and 3).
Figure 6:
In vivo metabolic labeling of
Hba1p. A, immunoprecipitates of S. pombe extracts
that had been labeled with [S]methionine. B, immunoprecipitates of S. pombe extracts that had
been labeled with
[
P]H
PO
. For both
experiments, S. pombe was transformed with pREP4 alone or
either hba1 or hba1-c-Myc under the control of the nmt1 promoter. Cultures were grown for 18 h in the absence of
thiamine to induce expression from the nmt1 promoter. The two
faster migrating bands in each of the hba1-c-Myc transformants
represent degradation products of Hba1p (data not
shown).
To further investigate the nature of the Hba1p doublet, we repeated
the metabolic labeling experiment with
[P]orthophosphate to determine whether the
observed heterogeneity was due to post-translational modification such
as phosphorylation. Derepressed strains transformed with vector alone,
Hba1, or Hba1-c-Myc were metabolically labeled for 3 h with
[
P]orthophosphate, immunoprecipitated, and
analyzed by SDS-PAGE as before. In vector alone and Hhba1-transformed
cells, no signal was detected. In contrast, a single phosphoprotein of
approximately 70 kDa was detected in addition to the two lower
molecular mass labeled proteolytic products of 28 and 30 kDa (Fig. 6B). These results demonstrate that Hba1 is a
nuclear phosphoprotein.
To determine if overexpression of hba1 affected message levels of any of the known genes conferring BFA resistance, Northern analysis was performed using total RNA isolated from a wild type strain transformed with a multicopy plasmid containing hba1 (pBAR2-1H) or vector alone. The resulting blot was hybridized with probes for hba1, hba2, crm1, pap1, and leu1a as a control for loading. As shown in Fig. 7, a weak hba1 signal could be detected only in the strain overexpressing this gene from the high copy plasmid pHBA1 (pBAR2-1H). No change in either hba2, crm1, or pap1 was detected in the strain overexpressing hba1. These results suggest that BFA resistance conferred by hba1 is independent of the crm1 regulatory pathway.
Figure 7: Overexpression expression of hba1 and its effects on other BFA resistance conferring genes. Ten micrograms of total RNA isolated from wild type S. pombe transformed with either pFL20 or pHBA1 (pBAR2-1H). RNA was fractionated on a 1% agarose gel and transferred to nitrocellulose. The filter was hybridized with a probe specific for hba1, hba2, crm1, pap1, and leu1a.
In this report we describe the characterization of hba1, a S. pombe gene conferring brefeldin A resistance. In the initial screen for genes conferring BFA resistance, plasmids encoding hba1 were the second most frequently isolated clones (Turi et al., 1994), suggesting that overexpression of Hba1p is an efficient mechanism of resistance. Plasmids containing hba1 confer high levels of BFA resistance in a growth assay and only moderate levels of resistance in a secretion assay. This difference in the levels of resistance presumably reflects the transient nature of the secretion assay, i.e. examination of resistance within a 30-min period versus extended periods (several hours).
The hba1 gene encodes a nuclear protein essential for S. pombe viability. Initial characterization of Hba1p yielded several unexpected results. Although the Hba1-c-Myc fusion protein was expected to migrate with a mobility of 41 kDa, the observed mobility for this protein was approximately 70 kDa. The aberrant mobility of Hba1p can be partially explained by its charged nature. At pH 7.0, 134 of the 399 amino acids are charged yielding a net overall charge of -4.2 (pI = 5.9).
A second
possible contributing factor to the aberrant mobility of Hba1p is
posttranslational modification. Immunopreciptation of the c-Myc
epitope-tagged Hba1p after [S]methionine
labeling yielded a broad heterogeneous band. Labeling with
[
P]orthophosphate determined Hba1p to be a
phosphoprotein.
A clue to the function of Hba1p may reside in its homology to proteins that interact with the nuclear low molecular weight GTP-binding protein Ran. This GTP-binding protein and its exchange factor, RCC1, have been implicated in several nuclear functions including nuclear import and export, regulation of the cell cycle, and maintenance of chromatin and nuclear structure (Dasso, 1993; Sazer and Nurse, 1994; Demeter et al., 1995). The hba1 gene product contains a 57-amino acid segment that is highly conserved in Ran-binding proteins (RanBPs), as well as a protein of the nuclear pore complex, and several other proteins of unknown function (Hartmann and Görlich, 1995). RanBP1 has been demonstrated to maintain Ran in the GTP-bound state or to act as a costimulator of GTP hydrolysis depending on the absence or presence of Ran-GTPase activating protein (Bischoff et al., 1995). Mutation of the first conserved glutamine, analogous to residue 266 of Hba1p, has been shown to reduce the binding of RanBP1 to Ran, suggesting a critical role for this domain in protein-protein interaction (Beddow et al., 1995).
The S. cerevisiae genes GSP1, PRP20, and YRB1 encode
homologues of Ran, RCC1, and RanBP1. In the fission yeast S.
pombe, spi1 and pim1 encode homologues of Ran
and RCC1. A Spi1-binding protein (Sbp1) with homology to RanBP1 has
recently been identified. ()(
)This leaves the
possibility that hba1 encodes a second Spi1-binding protein.
However, using a yeast two-hybrid system with Hba1p and Spi1p, we have
not been able to detect interaction between the two proteins. (
)Additional evidence that Hba1p may not interact with Spi1p
is supported by the results of an experiment in which
Spi1p[
P]GTP was used to probe yeast cell
extracts. No interacting proteins were identified with a molecular mass
similar to that predicted for Hba1 (Coutavas et al., 1993), (
)yet this protein overlay assay was able to detect an
interaction with S. cerevisiae RanBP (YRB1)
(Ouspenski et al. 1995). It is possible that Hba1p interacts
with a different as yet unidentified S. pombe nuclear GTPase
or that the interaction with Spi1p may not have been detected using the
two-hybrid system for technical reasons or by the protein overlay due
to low level expression or the inability of the protein to renature
into its active conformation.
Previously we have identified two additional S. pombe nuclear proteins, encoded by crm1 and pap1, which are capable of conferring BFA resistance (Turi et al., 1994). The pap1 gene encodes the S. pombe homologue of the mammalian AP1 transcription factor (Toda et al., 1991). Transcriptional regulation by pap1 is negatively regulated by the gene product of crm1 (Toda et al., 1992; Shimanuki et al., 1995). Because these two proteins regulate transcription of other genes responsible for conferring drug resistance, we determined if these genes affected hba1 transcription and also if overexpression of hba1 effected transcription of known genes involved in drug resistance. Neither crm1 or pap1 effected hba1 expression. Of the known genes capable of conferring BFA resistance when overexpressed, expression of these markers was not increased in the presence of elevated levels of hba1. Indeed, expression of hba2, a multidrug resistance transporter capable of conferring BFA resistance, was decreased in cells containing elevated levels of hba1. These results suggest that hba1 mediates BFA resistance via an undefined pathway. While the exact mechanism by which hba1 confers BFA resistance is unclear, the isolation of this gene which encodes a nuclear protein belonging to the RanBP family of proteins, has potentially expanded the role of RanBP-like proteins. Thus, through a combined genetic and biochemical approach, one may identify both the cellular function of hba1 and determine its role in BFA resistance.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U38783[GenBank].