Biochimica et Biophysica Acta, 1156 (1992) 1-6
1
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4165/92/$05.00
BBAGEN 23736
Isolation, partial amino acid sequence, and cellular distribution of heat-shock protein hsp98 from Neurospora crassa Alexei O. Vassilev a, Nora Plesofsky-Vig a,b and R o b e r t Brambl a,c a Department of Plant Biology, b Department of Genetics and Cell Biology, and c Plant Molecular Genetics Institute, The University of Minnesota, Saint Paul, MN (USA)
(Received 29 June 1992)
Key words: Heat-shock protein; Protein isolation; Amino acid sequence; Cellular distribution; (N. crassa) Hsp98 is one of the most prominent proteins synthesized during the heat-shock response of Neurospora crassa. We purified hsp98 and determined the amino acid sequence of two overlapping peptides obtained by cyanogen bromide cleavage. This 28 amino acid sequence from hsp98 has 75% homology with a region of the CIpB protein of Escherichia coli and 86% homology to a 96-kDa protein of Trypanosoma brucei. It also has 71% homology to hspl04 of Saccharomyces cerevisiae. Hsp98 was enriched in the microsomal fraction of heat-shocked cells. Sucrose gradient analysis of this cellular fraction showed that the three major high molecular weight heat-shock proteins (hsp98, 83 and 67) were more concentrated in polyribosomes than in monoribosomes. Another newly synthesized protein, p28, was strongly enriched in monoribosomes. After dissociation of the polyribosomes into ribosomal subunits, the three major heat-shock proteins were shown to be localized preferentially in the large subunit. Whereas p28 was also strongly associated with the large ribosomal subunit, a newly synthesized protein of about 22 kDa was exclusively associated with the small subunit.
Introduction When exposed to supra-optimal temperatures, all types of organisms display a heat-shock response which increases their tolerance to heat stress. This response involves the induction of synthesis of a group of heatshock proteins and repression of synthesi s of normal proteins. One major goal of studying the heat shock response is to determine how these proteins function within the cell, and a related objective is to determine the intracellular locations of the various heat shock proteins. Neurospora crassa synthesizes three major high molecular mass heat-shock proteins of 98, 83 and 67 kDa [1]. Heat-shock proteins of similar size are expressed by many organisms. Although the hsp83 and hsp70 families of proteins have been the best characterized, many organisms also synthesize a heat shock protein of approximately 100 kDa [2,3]. The 98 kDa
Correspondence to: Robert Brambl, Department of Plant Biology, 220 Biological Sciences Center, The University of Minnesota, Saint Paul, MN 55108 USA. Abbreviations: hsp, heat shock protein; PHT, phenylthiohydantoin; HPLC, high-performance liquid chromatography; FPLC, fast protein liquid chromatography; PIR, Protein Information Resource database of the National Biomedical Research Foundation.
protein of Neurospora is most highly expressed early in the heat-shock response, and its synthesis declines sooner than that of the other proteins during continuous heat-shock [4]. Three lower molecular weight heat shock proteins (hsp38, 34 and 30) are enriched in the mitochondrial fraction of Neurospora cells [4,5]. In this report we show that some proteins synthesized during heat shock are enriched in the microsomal fraction of heat-shocked cells of N. crassa. One of these proteins, hsp98, was purified and a partial amino acid sequence was obtained. This 28 amino acid sequence has 86% homology to a region within a 96 kDa protein of Trypanosoma brucei [6] and 71% homology to hspl04 of Saccharomyces cerecisiae [7]. These proteins are related to the ClpA protein of Escherichia coli which is a regulatory subunit of an ATP-dependent protease [6]. Materials and Methods Cell culture and extraction Conidia of N. crassa 74A were germinated at 30°C
as described earlier [4], and the heat-shock response was induced at 45°C. L-[35S]methionine ( 1 / x C i / m l ) was added to the culture 10 min after the beginning of heat shock, and 20 min later (1 min before harvest) a 1000-fold excess of unlabeled methionine was added to
the culture (to 1 /~M). Cells were collected by filtration, washed with fresh 4°C culture medium [8] containing unlabeled methionine and disrupted [4]. For purification of hsp98, cells were disrupted in standard protein buffer (10 mM triethanolamine, 10 mM KC1, 0.1 M NaCI, 5 mM MgC12, 1 mM EDTA, 6 mM /3-mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride, 10% glycerol [pH 7.8]), and for polyribosome isolation the cells were disrupted in buffer A [9]. The homogenate was centrifuged at 10 000 x g~v for 5 min, the supernatant was centrifuged at 30000 X g,v for 20 rain and the resulting supernatant was used for protein purification. To prepare a microsome fraction, this supernatant was centrifuged at 140000Xg,v for 90 rain and the pellet resuspended in buffer B [9] for subsequent isolation of polyribosomes.
Isolation of hsp98 Conidial cells, germinated for 5 h at 30°C, were heat-shocked (45°C) for 30 min (including a 20-min radiolabeling with L-[35S]methionine [0.1 /zCi/ml]. Cells (30 g) were disrupted and centrifuged, and the mitochondria-free extract was layered onto a 15 ml column containing phosphocellulose P l l (Whatman). Material not binding to this column was layered onto a 10-ml column of heparin-agarose (Pharmacia), washed with 10 vol of standard buffer and eluted with a linear gradient of 0.1-0.8 M NaC1 in standard buffer. The fractions containing radioactive material were examined by electrophoresis. Fractions with hsp98 were combined, diluted to 0.1 M NaCI and applied to a Mono Q column of a FPLC system (Pharmacia). The elution was performed with a linear gradient of 0.1-0.6 M NaC1 in standard buffer.
methanol-10% acetic acid, rinsed in water, dried, and stored at - 70°C. The sequence analysis was performed with a Porton 2090E gas-phase sequencer (Porton Instruments), using a Hewlett-Packard 1090 HPLC system with an AminoQuant column (2.1 × 200 ram) to separate the PTH-derivatized amino acids.
Sucrose gradient centrifugation Sucrose gradients were prepared as described earlier [9], with 0.2-0.5 mg of RNA (1 mg = 24 A260units) layered onto each gradient. After 3.5 h of centrifugation at 25000 rpm in a Beckman SW-28 rotor, gradients were fractionated automatically with an ISCO fractionator and a UV monitor [12]. For electrophoretic analysis of fractions obtained from the gradients, material was precipitated with 2-3 vol of cold ethanol for 20 h at - 2 0 ° C and then dissolved in electrophoresis sample buffer [11]. Following electrophoresis, the gel was fixed with 30% methanol-10% acidic acid, treated with Amplify (Amersham), dried, and exposed to X-ray film. To prepare ribosome subunits, fractions obtained from gradients were centrifuged at 110000 x g,,. for 20 h, resuspended in buffer
hsp98 hsp83 hsp67
1
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4165/92/$05.00
BBAGEN 23736
Isolation, partial amino acid sequence, and cellular distribution of heat-shock protein hsp98 from Neurospora crassa Alexei O. Vassilev a, Nora Plesofsky-Vig a,b and R o b e r t Brambl a,c a Department of Plant Biology, b Department of Genetics and Cell Biology, and c Plant Molecular Genetics Institute, The University of Minnesota, Saint Paul, MN (USA)
(Received 29 June 1992)
Key words: Heat-shock protein; Protein isolation; Amino acid sequence; Cellular distribution; (N. crassa) Hsp98 is one of the most prominent proteins synthesized during the heat-shock response of Neurospora crassa. We purified hsp98 and determined the amino acid sequence of two overlapping peptides obtained by cyanogen bromide cleavage. This 28 amino acid sequence from hsp98 has 75% homology with a region of the CIpB protein of Escherichia coli and 86% homology to a 96-kDa protein of Trypanosoma brucei. It also has 71% homology to hspl04 of Saccharomyces cerevisiae. Hsp98 was enriched in the microsomal fraction of heat-shocked cells. Sucrose gradient analysis of this cellular fraction showed that the three major high molecular weight heat-shock proteins (hsp98, 83 and 67) were more concentrated in polyribosomes than in monoribosomes. Another newly synthesized protein, p28, was strongly enriched in monoribosomes. After dissociation of the polyribosomes into ribosomal subunits, the three major heat-shock proteins were shown to be localized preferentially in the large subunit. Whereas p28 was also strongly associated with the large ribosomal subunit, a newly synthesized protein of about 22 kDa was exclusively associated with the small subunit.
Introduction When exposed to supra-optimal temperatures, all types of organisms display a heat-shock response which increases their tolerance to heat stress. This response involves the induction of synthesis of a group of heatshock proteins and repression of synthesi s of normal proteins. One major goal of studying the heat shock response is to determine how these proteins function within the cell, and a related objective is to determine the intracellular locations of the various heat shock proteins. Neurospora crassa synthesizes three major high molecular mass heat-shock proteins of 98, 83 and 67 kDa [1]. Heat-shock proteins of similar size are expressed by many organisms. Although the hsp83 and hsp70 families of proteins have been the best characterized, many organisms also synthesize a heat shock protein of approximately 100 kDa [2,3]. The 98 kDa
Correspondence to: Robert Brambl, Department of Plant Biology, 220 Biological Sciences Center, The University of Minnesota, Saint Paul, MN 55108 USA. Abbreviations: hsp, heat shock protein; PHT, phenylthiohydantoin; HPLC, high-performance liquid chromatography; FPLC, fast protein liquid chromatography; PIR, Protein Information Resource database of the National Biomedical Research Foundation.
protein of Neurospora is most highly expressed early in the heat-shock response, and its synthesis declines sooner than that of the other proteins during continuous heat-shock [4]. Three lower molecular weight heat shock proteins (hsp38, 34 and 30) are enriched in the mitochondrial fraction of Neurospora cells [4,5]. In this report we show that some proteins synthesized during heat shock are enriched in the microsomal fraction of heat-shocked cells of N. crassa. One of these proteins, hsp98, was purified and a partial amino acid sequence was obtained. This 28 amino acid sequence has 86% homology to a region within a 96 kDa protein of Trypanosoma brucei [6] and 71% homology to hspl04 of Saccharomyces cerecisiae [7]. These proteins are related to the ClpA protein of Escherichia coli which is a regulatory subunit of an ATP-dependent protease [6]. Materials and Methods Cell culture and extraction Conidia of N. crassa 74A were germinated at 30°C
as described earlier [4], and the heat-shock response was induced at 45°C. L-[35S]methionine ( 1 / x C i / m l ) was added to the culture 10 min after the beginning of heat shock, and 20 min later (1 min before harvest) a 1000-fold excess of unlabeled methionine was added to
the culture (to 1 /~M). Cells were collected by filtration, washed with fresh 4°C culture medium [8] containing unlabeled methionine and disrupted [4]. For purification of hsp98, cells were disrupted in standard protein buffer (10 mM triethanolamine, 10 mM KC1, 0.1 M NaCI, 5 mM MgC12, 1 mM EDTA, 6 mM /3-mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride, 10% glycerol [pH 7.8]), and for polyribosome isolation the cells were disrupted in buffer A [9]. The homogenate was centrifuged at 10 000 x g~v for 5 min, the supernatant was centrifuged at 30000 X g,v for 20 rain and the resulting supernatant was used for protein purification. To prepare a microsome fraction, this supernatant was centrifuged at 140000Xg,v for 90 rain and the pellet resuspended in buffer B [9] for subsequent isolation of polyribosomes.
Isolation of hsp98 Conidial cells, germinated for 5 h at 30°C, were heat-shocked (45°C) for 30 min (including a 20-min radiolabeling with L-[35S]methionine [0.1 /zCi/ml]. Cells (30 g) were disrupted and centrifuged, and the mitochondria-free extract was layered onto a 15 ml column containing phosphocellulose P l l (Whatman). Material not binding to this column was layered onto a 10-ml column of heparin-agarose (Pharmacia), washed with 10 vol of standard buffer and eluted with a linear gradient of 0.1-0.8 M NaC1 in standard buffer. The fractions containing radioactive material were examined by electrophoresis. Fractions with hsp98 were combined, diluted to 0.1 M NaCI and applied to a Mono Q column of a FPLC system (Pharmacia). The elution was performed with a linear gradient of 0.1-0.6 M NaC1 in standard buffer.
methanol-10% acetic acid, rinsed in water, dried, and stored at - 70°C. The sequence analysis was performed with a Porton 2090E gas-phase sequencer (Porton Instruments), using a Hewlett-Packard 1090 HPLC system with an AminoQuant column (2.1 × 200 ram) to separate the PTH-derivatized amino acids.
Sucrose gradient centrifugation Sucrose gradients were prepared as described earlier [9], with 0.2-0.5 mg of RNA (1 mg = 24 A260units) layered onto each gradient. After 3.5 h of centrifugation at 25000 rpm in a Beckman SW-28 rotor, gradients were fractionated automatically with an ISCO fractionator and a UV monitor [12]. For electrophoretic analysis of fractions obtained from the gradients, material was precipitated with 2-3 vol of cold ethanol for 20 h at - 2 0 ° C and then dissolved in electrophoresis sample buffer [11]. Following electrophoresis, the gel was fixed with 30% methanol-10% acidic acid, treated with Amplify (Amersham), dried, and exposed to X-ray film. To prepare ribosome subunits, fractions obtained from gradients were centrifuged at 110000 x g,,. for 20 h, resuspended in buffer
hsp98 hsp83 hsp67
