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Identification of a processed protein related to the human chaperonins (hsp 60) protein in mammalian kidney
BIOCHEMICAL
Vol. 185, No. 2, 1992
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages
June 15, 1992
IDENTIFTCATION HUMAN
OF A PROCl3SSED PROTEIN
CHAPERONINS
(HSP 60) PROTEIN
Willie R. Ross, William
RELATED
633-687
To THE
IN MAMMALTANKIDNEY
S. Bertrand and Aubrey R. Morrison
Washington University School of Medicine Departments of Medicine and Molecular Biology and Pharmacology St. Louis, Missouri 63110 Received
April
24,
1992
The chaperonin family of proteins, which includes GroEL protein of E. coli, yeast heat shock protein (hsp-60) and the ribulose-1-5-bisphosphate carboxylase (Rubis Co.) subunit binding protein of plant choloroplasts, shows strong sequence homology to the Chinese hamster ovary (CHO) mitochondrial Pl protein. We have identified a 60 kDa protein from bovine kidney which by N-terminal sequencing gives the amino acid sequence AKDVKFGADARALLMLQGVDLLADA. Bovine whole kidney membranes were delipidated, solubilized with octyl glucoside and fractionated over an affinity column using the amiloride analog 5-N pyrazine amiloride as the ligand. After extensive washing with 200 mM NaCl, the column was eluted with pH 4.0 buffer. Analysis of column fractions on a 7.5 % polyacrylamide gel revealed 3-4 bands with a predominant band at 60,000 Da. Amino acid analysis after transfer to immobilon membranes demonstrated sequence identity to the human HSP (60), extending 24 amino acids from the N-terminus, but lacking the leader sequence. These data indicate that a processed form of a protein related to the human HSP (60) chaperonin is associated with a membrane fraction in the mammalian kidney, and that the processed form of the protein binds strongly to an amiloride affinity support. 0 1992 Academic Press, Inc.
The complex cellular processes leading to proper folding, assembly and transport of nascent polypeptides have only recently been elaborated (l-2). denaturation of a highly compact intermediate intracellular
Protein folding in viva involves
structure in an equally compact concentrated
matrix (3-4). A highly evolutionary conserved class of proteins termed molecular
chaperonins recognize structural motifs inherent in proteins destined for processing and secretion (5-7).
Through poorly understood interactions, chaperonins function in an ATP-dependent
manner to facilitate correct post-translational
assembly and transport of certain oligomeric
proteins (8). By definition, chaperonins are not part of the assembled complex (9). Members of the chaperonin class of proteins included the Gro-EL protein of E.coIi (6, lo), which is involved in bacteriophages lambda and T4 head morphogenesis, rubisco (ribulose 15 bisphosphate carboxylase; RBUp2) subunit binding protein (RBUp2BP) (1, 11,12) which functions in the assembly of rub&o or Pl in mammalian
of plant chloroplasts
in plant chloroplasts, and hsp 60 in yeast
cells (13-15) - a protein found in eukaryotic cell mitochondria 0006-291X/92
683
which is $4.00
Copyright 0 1992 by Academic Press, Inc. AI1 righrs of reproducrion in any form reserved.
Vol.
185,
No.
BIOCHEMICAL
2, 1992
AND
required for import of protein into mitochondria
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
as well as their assembly into oligomeric
mitochondrial enzyme complexes. A cDNA of the mitochondrial
Pl encodes a protein of 60,983
Da and includes a putative 26 amino acid presequence at its N-terminus (15 .) Similar to the related bacterial protein Gro-EL, the Pl protein exists as a homo-oligomeric complex of seven subunits.
Electron microscopy of Gro-EL particles followed by 3-D
reconstruction indicates a polar channel and possibly a partially buried area for hydrophobic contacts (16). The presence of a putative hydrophobic core, as well as the high content of hydrophobic amino acids in the Gro-EL and Pl sequences offers the potential for isolating the protein
by a number
of chromatographic
methods, including
hydrophobic
interaction
chromatography and affinity chromatography with stable hydrophobic ligands. Our laboratory has developed a number of amiloride analogs with hydrophobic substituents on the 5-amino nitrogen atom of the pyrazine ring (17).
We describe in this manuscript the ability of an
amiloride analog to bind a mitochondrial Pl-like MATERIALS
protein from bovine renal tissue.
AND METHODS
Bovine kidneys were obtained fresh from a local slaughterhouse. Whole kidneys were delipidated by acetone/hexane extraction - 260 gm of kidney was homogenized in acetone, filtered under vacuum, then washed three times with cold acetone. The protein was then washed three times with hexane, filtered under vacuum and lyophylized. The 31 gm of delipidated membranes were solubilized in 5% Triton X-100, 100 mM KCl, 50 mM mannitol, 40 mM MES, 5% glycerol, 1 mM PMSF, and 10 rig/ml of pepstatin and leupeptin, pH 7.4 for 30 min at 4”C, then centrifuged at 105,000 x g for 60 min. Solubilized protein, 200 ng was fractionated over an affinity column using 5-N-pyrazine amiloride (PZA) as the immpbilized ligand. The affinity column was prepared by coupling 25 ng of 5-N-PZA to 5 gm activated CH sepharose 4B (previously swollen in 1 N HCl) in 0.1 M NaH03, 0.5 M NaCl for 60 min at 37°C. Excess active sites were blocked with 1 M ethanolamine. The coupled matrix was poured into a glass Econocolumn. After protein loading the affinity column was washed with 20 bed volumes of 200 mM NaCl pH 7.4, then eluted with 20 bed volumes at 200 mM NaCl, 0.1% Triton X-100, 10 mM Hepes, pH 4.0. Protein concentration in the column eluates was determined by the BioRad protein assay. Aliquots were dissolved in SDS sample buffer containing 5% P-mercaptethanol, boiled at 100°C for 5 min, then analyzed over a 7.5% polyacrylamide gel. The gel was transferred to Immobilon membranes using a Transphor apparatus for 1 hr at 37”C, according to the method of Towbin et al (18). The membrane was stained with 0.1% Coomassie Blue R250 in 50% methanol, then destained with 50% methanol. The 60 kDa protein was excised and submitted for amino acid analysis at the Washington University protein chemistry laboratory. RESULTS The polyacrylamide
AND DISCUSSION
gel analysis of the PZA column eluate, pH 4.0, is shown in Figure
1. The predominant species, a polypeptide of 60,000 Da, bound avidly to the PZA affinity column and could only be eluted by pH4 buffered saline. Amino acid analysis of the 60 kDa 684
Vol.
BIOCHEMICAL
185, No. 2, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Figure 1. SDS PAGE of pH 4.0 eluateof PZA affinity column.
protein was obtained (Figure 2). Only the first 24 amino acids from the NH2 terminus are shown. These 24 amino acids showed striking sequences homology to the coding region of the human chaperonin hsp60 (14). Of note, the 26 amino acid presequence found in the cDNA for human hsp 60 was not present, indicating that the mature processed form of the protein had been isolated. The N-terminal presequence has been shown to form a strongly amphiphilic alpha-helix which promotes protein translocation into mitochondria (19). Singh et. al. observed that in the absence of the N-terminal
sequence in cDNA constructs no import into mitochondria occurred.
It is reasonable to postulate the N-terminal
sequence of the Pl protein isolated from the PZA
column was cleaved after translocation to the appropriate subcellular compartment. A number of laboratories have successfully cloned and sequenced mammalian related proteins (14, 15) (20-21).
Pl and
We report in this manusript the isolation of a chaperonin
protein using conventional affinity techniques. The diuretic amiloride was an unlikely candidate for an affinity ligand.
Amiloride
is a pyrazinoyl guanidine bearing amino groups on the 3 and
5 position of the pyrazine ring (22). Amiloride and its analogs are felt to be specific inhibitors of a few ion transport systems including the Na+/H+ and Na+/C!a2+
antiporter, the epithelial sodium channel,
exchanger, but however inhibit a number of membrane transport processes and
enzymes (23-24). Since the initial description of amiloride inhibition
of the Na+/H+
exchanger, various
analogs have been developed which were felt to have greater specificity for inhibition Na+/H+
of
exchange. In general, amiloride analogs with hydrophobic groups on the 5-amino
nitrogen atom are more potent and specific for the Na+/H+
exchanger (25-26).
Two
hydrophobic substituents offer higher potency and specificity than one, and in several cell lines, amiloride analogs exhibit IC50 values of Na+/H+ exchange less than 100 nM. Coincident with 685
Vol.
185,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AKDVKFGADARALMLQGVDLLADA
Figure 2. NH2-terminal sequenceof 60 kDa protein obtained from SDS-PAGE.
the increased potency of these analogs is a greater propensity
interactions with adjacent proteins and cellular matrix.
for nonspecific hydrophobic
The hydrophobic nature of amiloride
analogs may be exploited for use in isolating proteins whose biologic activities might be adversely affected by HPLC hydrophobic interaction columns.
The amiloride
analog 5-N-Pyrazine
amiloride proved to be effective in a single step isolation of the Pl protein from bovine renal tissue. Immunofluorescence mammalian
cells (14).
studies with Pl antibody have shown staining of mitochondria in
The presence of a Pl related protein in bovine renal tissue is in
accordance with the ubiquitous distribution
of molecular chaperonins.
The high degree of
sequence homology between the isolated renal protein, the human Pl, as well as the Gro-EL protein further supports the premise that these highly evolutionary protein perform similar functions in different species (6,8,14,15).
Although our preparation of total bovine kidney
membranes precludes analysis of a specific mitochondrial fraction, it is likely that the 60 kda Pl related protein is mitochondrial
in origin.
In addition, it is clear that the processed protein is
membrane associated. Acknowledgment.
This work was supported by NIH Public Health Award DK 38111.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Gierasch, L.M. and King, J. (1989) Protein Folding: Deciphering the Second Half of the Genetic Code. American Association for the Advancement of Science. Fischer, G. and Schmid, F.K. (1990) Biochemistry 29, 22052212. Bychkova, V.E., Pain, R.H. and Ptitsyn, O.B. (1988) FEBS Lett. 238, 231-234. Kuwajina, K. (1987) Prot. Struct. Funct. Genet. 3, 87-103. Ellis, J. (1987) Nature 328, 378-379. Hemmingsen, SM., Woolford, C., van der Vies, S.M, Tilly, K.. Dennis, D.T., Georgopoulos, C.P., Hendrix, R.W., and Ellis, R.J. (1988) Nature 333, 330-334. Ellis, R.J. and van der Vies, S.M. (1988) Photosynth. Res. 16, 101-115. Ellis, R.J., and Hemmingsen, S.M. (1988) Trends Biochem. Sci. 14, 339-342. Ellis, R.J., van der Vies, S.M. and Hemmingsen, S.M. (1989) B&hem. Sot. Symp. 55, 145-153. Goloubinoff, P., Christeller, J.T., Gatenby, A.A. and Lorimer, G.H. (1989) Nature 342, 884-888. Ellis, R.J. (1990) Seminars in Cell Biol. 1, l-9. Roy H. (1989) The Plant Cell. 1, 1035-1042. Cheng, M.Y., Hartl, F.V., Martin, J., Pollock, R.A., Kalousek, F., Neupert, W., Hallberg, E.M., Hallberg, R.L., and Horwich, A.L. (1989) Nature. 337, 620-625. Jindal, S.K., Dudani, A.K., Singh, B., Harley, C.B., and Gupta, R.S. (1989) Mol. Cell. Biol. 9, 2279-22893. 686
Vol.
15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
185,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Picketts, D.J., Mayanil, C.S.K. and Gupta, R.S. (1989) J. Biol. Chem. 264, 1200112008. Carrascosa, J.L., Abella, G., Marco, S., and Carazo, J.M. (1990) J. Struct. Biol. 104, 2-8. Ross, W.R., Betrand W.S., and Morrison, A.R. (1990) J. Biol. Chem 265,5341-5344. Towbin, H., Stachelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. Singh, B., Patel, H.V., Ridley, R.G., Freeman, K.B. and Gupta, R.S. (1990) B&hem. Biophys. Res. Comm. 169, 391-396. Reading, D.S., Hallberg, R.C., and Myers, A.M. (1989) Nature. 337, 655-659. Peralta, D., Hartman, D.J., McIntosh, A.M., Hoogenraad, N.J., and Hoj, P.B. (1990) Nucl. Acids Rex 18, 7162. Cragoe, E.J. Jr., Woltersdorf, 0-W. Jr., Bicking, J.B., Kwong, S.F., and Jones, J.H. (1967) J. Med. Chem. 10, 66-75. Benos, D.J. (1982) Am. J. Physiol. 242:C131-C145. Kleyman, T.R., Cragoe, E.J. Jr. (1988) J. Membrane Biol. 105, 1-21. Simchowitz, L., Cragoe, E.J., Jr. (1986) Mol. Pharmnacol. 30, 112-120. Simchowitz, L., Woltersdorf, O.W., Cragoe, E.J. Jr. (1987) J. Biol. Chem. 262, 1587515975.
687
Vol. 185, No. 2, 1992
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages
June 15, 1992
IDENTIFTCATION HUMAN
OF A PROCl3SSED PROTEIN
CHAPERONINS
(HSP 60) PROTEIN
Willie R. Ross, William
RELATED
633-687
To THE
IN MAMMALTANKIDNEY
S. Bertrand and Aubrey R. Morrison
Washington University School of Medicine Departments of Medicine and Molecular Biology and Pharmacology St. Louis, Missouri 63110 Received
April
24,
1992
The chaperonin family of proteins, which includes GroEL protein of E. coli, yeast heat shock protein (hsp-60) and the ribulose-1-5-bisphosphate carboxylase (Rubis Co.) subunit binding protein of plant choloroplasts, shows strong sequence homology to the Chinese hamster ovary (CHO) mitochondrial Pl protein. We have identified a 60 kDa protein from bovine kidney which by N-terminal sequencing gives the amino acid sequence AKDVKFGADARALLMLQGVDLLADA. Bovine whole kidney membranes were delipidated, solubilized with octyl glucoside and fractionated over an affinity column using the amiloride analog 5-N pyrazine amiloride as the ligand. After extensive washing with 200 mM NaCl, the column was eluted with pH 4.0 buffer. Analysis of column fractions on a 7.5 % polyacrylamide gel revealed 3-4 bands with a predominant band at 60,000 Da. Amino acid analysis after transfer to immobilon membranes demonstrated sequence identity to the human HSP (60), extending 24 amino acids from the N-terminus, but lacking the leader sequence. These data indicate that a processed form of a protein related to the human HSP (60) chaperonin is associated with a membrane fraction in the mammalian kidney, and that the processed form of the protein binds strongly to an amiloride affinity support. 0 1992 Academic Press, Inc.
The complex cellular processes leading to proper folding, assembly and transport of nascent polypeptides have only recently been elaborated (l-2). denaturation of a highly compact intermediate intracellular
Protein folding in viva involves
structure in an equally compact concentrated
matrix (3-4). A highly evolutionary conserved class of proteins termed molecular
chaperonins recognize structural motifs inherent in proteins destined for processing and secretion (5-7).
Through poorly understood interactions, chaperonins function in an ATP-dependent
manner to facilitate correct post-translational
assembly and transport of certain oligomeric
proteins (8). By definition, chaperonins are not part of the assembled complex (9). Members of the chaperonin class of proteins included the Gro-EL protein of E.coIi (6, lo), which is involved in bacteriophages lambda and T4 head morphogenesis, rubisco (ribulose 15 bisphosphate carboxylase; RBUp2) subunit binding protein (RBUp2BP) (1, 11,12) which functions in the assembly of rub&o or Pl in mammalian
of plant chloroplasts
in plant chloroplasts, and hsp 60 in yeast
cells (13-15) - a protein found in eukaryotic cell mitochondria 0006-291X/92
683
which is $4.00
Copyright 0 1992 by Academic Press, Inc. AI1 righrs of reproducrion in any form reserved.
Vol.
185,
No.
BIOCHEMICAL
2, 1992
AND
required for import of protein into mitochondria
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
as well as their assembly into oligomeric
mitochondrial enzyme complexes. A cDNA of the mitochondrial
Pl encodes a protein of 60,983
Da and includes a putative 26 amino acid presequence at its N-terminus (15 .) Similar to the related bacterial protein Gro-EL, the Pl protein exists as a homo-oligomeric complex of seven subunits.
Electron microscopy of Gro-EL particles followed by 3-D
reconstruction indicates a polar channel and possibly a partially buried area for hydrophobic contacts (16). The presence of a putative hydrophobic core, as well as the high content of hydrophobic amino acids in the Gro-EL and Pl sequences offers the potential for isolating the protein
by a number
of chromatographic
methods, including
hydrophobic
interaction
chromatography and affinity chromatography with stable hydrophobic ligands. Our laboratory has developed a number of amiloride analogs with hydrophobic substituents on the 5-amino nitrogen atom of the pyrazine ring (17).
We describe in this manuscript the ability of an
amiloride analog to bind a mitochondrial Pl-like MATERIALS
protein from bovine renal tissue.
AND METHODS
Bovine kidneys were obtained fresh from a local slaughterhouse. Whole kidneys were delipidated by acetone/hexane extraction - 260 gm of kidney was homogenized in acetone, filtered under vacuum, then washed three times with cold acetone. The protein was then washed three times with hexane, filtered under vacuum and lyophylized. The 31 gm of delipidated membranes were solubilized in 5% Triton X-100, 100 mM KCl, 50 mM mannitol, 40 mM MES, 5% glycerol, 1 mM PMSF, and 10 rig/ml of pepstatin and leupeptin, pH 7.4 for 30 min at 4”C, then centrifuged at 105,000 x g for 60 min. Solubilized protein, 200 ng was fractionated over an affinity column using 5-N-pyrazine amiloride (PZA) as the immpbilized ligand. The affinity column was prepared by coupling 25 ng of 5-N-PZA to 5 gm activated CH sepharose 4B (previously swollen in 1 N HCl) in 0.1 M NaH03, 0.5 M NaCl for 60 min at 37°C. Excess active sites were blocked with 1 M ethanolamine. The coupled matrix was poured into a glass Econocolumn. After protein loading the affinity column was washed with 20 bed volumes of 200 mM NaCl pH 7.4, then eluted with 20 bed volumes at 200 mM NaCl, 0.1% Triton X-100, 10 mM Hepes, pH 4.0. Protein concentration in the column eluates was determined by the BioRad protein assay. Aliquots were dissolved in SDS sample buffer containing 5% P-mercaptethanol, boiled at 100°C for 5 min, then analyzed over a 7.5% polyacrylamide gel. The gel was transferred to Immobilon membranes using a Transphor apparatus for 1 hr at 37”C, according to the method of Towbin et al (18). The membrane was stained with 0.1% Coomassie Blue R250 in 50% methanol, then destained with 50% methanol. The 60 kDa protein was excised and submitted for amino acid analysis at the Washington University protein chemistry laboratory. RESULTS The polyacrylamide
AND DISCUSSION
gel analysis of the PZA column eluate, pH 4.0, is shown in Figure
1. The predominant species, a polypeptide of 60,000 Da, bound avidly to the PZA affinity column and could only be eluted by pH4 buffered saline. Amino acid analysis of the 60 kDa 684
Vol.
BIOCHEMICAL
185, No. 2, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Figure 1. SDS PAGE of pH 4.0 eluateof PZA affinity column.
protein was obtained (Figure 2). Only the first 24 amino acids from the NH2 terminus are shown. These 24 amino acids showed striking sequences homology to the coding region of the human chaperonin hsp60 (14). Of note, the 26 amino acid presequence found in the cDNA for human hsp 60 was not present, indicating that the mature processed form of the protein had been isolated. The N-terminal presequence has been shown to form a strongly amphiphilic alpha-helix which promotes protein translocation into mitochondria (19). Singh et. al. observed that in the absence of the N-terminal
sequence in cDNA constructs no import into mitochondria occurred.
It is reasonable to postulate the N-terminal
sequence of the Pl protein isolated from the PZA
column was cleaved after translocation to the appropriate subcellular compartment. A number of laboratories have successfully cloned and sequenced mammalian related proteins (14, 15) (20-21).
Pl and
We report in this manusript the isolation of a chaperonin
protein using conventional affinity techniques. The diuretic amiloride was an unlikely candidate for an affinity ligand.
Amiloride
is a pyrazinoyl guanidine bearing amino groups on the 3 and
5 position of the pyrazine ring (22). Amiloride and its analogs are felt to be specific inhibitors of a few ion transport systems including the Na+/H+ and Na+/C!a2+
antiporter, the epithelial sodium channel,
exchanger, but however inhibit a number of membrane transport processes and
enzymes (23-24). Since the initial description of amiloride inhibition
of the Na+/H+
exchanger, various
analogs have been developed which were felt to have greater specificity for inhibition Na+/H+
of
exchange. In general, amiloride analogs with hydrophobic groups on the 5-amino
nitrogen atom are more potent and specific for the Na+/H+
exchanger (25-26).
Two
hydrophobic substituents offer higher potency and specificity than one, and in several cell lines, amiloride analogs exhibit IC50 values of Na+/H+ exchange less than 100 nM. Coincident with 685
Vol.
185,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AKDVKFGADARALMLQGVDLLADA
Figure 2. NH2-terminal sequenceof 60 kDa protein obtained from SDS-PAGE.
the increased potency of these analogs is a greater propensity
interactions with adjacent proteins and cellular matrix.
for nonspecific hydrophobic
The hydrophobic nature of amiloride
analogs may be exploited for use in isolating proteins whose biologic activities might be adversely affected by HPLC hydrophobic interaction columns.
The amiloride
analog 5-N-Pyrazine
amiloride proved to be effective in a single step isolation of the Pl protein from bovine renal tissue. Immunofluorescence mammalian
cells (14).
studies with Pl antibody have shown staining of mitochondria in
The presence of a Pl related protein in bovine renal tissue is in
accordance with the ubiquitous distribution
of molecular chaperonins.
The high degree of
sequence homology between the isolated renal protein, the human Pl, as well as the Gro-EL protein further supports the premise that these highly evolutionary protein perform similar functions in different species (6,8,14,15).
Although our preparation of total bovine kidney
membranes precludes analysis of a specific mitochondrial fraction, it is likely that the 60 kda Pl related protein is mitochondrial
in origin.
In addition, it is clear that the processed protein is
membrane associated. Acknowledgment.
This work was supported by NIH Public Health Award DK 38111.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Gierasch, L.M. and King, J. (1989) Protein Folding: Deciphering the Second Half of the Genetic Code. American Association for the Advancement of Science. Fischer, G. and Schmid, F.K. (1990) Biochemistry 29, 22052212. Bychkova, V.E., Pain, R.H. and Ptitsyn, O.B. (1988) FEBS Lett. 238, 231-234. Kuwajina, K. (1987) Prot. Struct. Funct. Genet. 3, 87-103. Ellis, J. (1987) Nature 328, 378-379. Hemmingsen, SM., Woolford, C., van der Vies, S.M, Tilly, K.. Dennis, D.T., Georgopoulos, C.P., Hendrix, R.W., and Ellis, R.J. (1988) Nature 333, 330-334. Ellis, R.J. and van der Vies, S.M. (1988) Photosynth. Res. 16, 101-115. Ellis, R.J., and Hemmingsen, S.M. (1988) Trends Biochem. Sci. 14, 339-342. Ellis, R.J., van der Vies, S.M. and Hemmingsen, S.M. (1989) B&hem. Sot. Symp. 55, 145-153. Goloubinoff, P., Christeller, J.T., Gatenby, A.A. and Lorimer, G.H. (1989) Nature 342, 884-888. Ellis, R.J. (1990) Seminars in Cell Biol. 1, l-9. Roy H. (1989) The Plant Cell. 1, 1035-1042. Cheng, M.Y., Hartl, F.V., Martin, J., Pollock, R.A., Kalousek, F., Neupert, W., Hallberg, E.M., Hallberg, R.L., and Horwich, A.L. (1989) Nature. 337, 620-625. Jindal, S.K., Dudani, A.K., Singh, B., Harley, C.B., and Gupta, R.S. (1989) Mol. Cell. Biol. 9, 2279-22893. 686
Vol.
15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
185,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Picketts, D.J., Mayanil, C.S.K. and Gupta, R.S. (1989) J. Biol. Chem. 264, 1200112008. Carrascosa, J.L., Abella, G., Marco, S., and Carazo, J.M. (1990) J. Struct. Biol. 104, 2-8. Ross, W.R., Betrand W.S., and Morrison, A.R. (1990) J. Biol. Chem 265,5341-5344. Towbin, H., Stachelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. Singh, B., Patel, H.V., Ridley, R.G., Freeman, K.B. and Gupta, R.S. (1990) B&hem. Biophys. Res. Comm. 169, 391-396. Reading, D.S., Hallberg, R.C., and Myers, A.M. (1989) Nature. 337, 655-659. Peralta, D., Hartman, D.J., McIntosh, A.M., Hoogenraad, N.J., and Hoj, P.B. (1990) Nucl. Acids Rex 18, 7162. Cragoe, E.J. Jr., Woltersdorf, 0-W. Jr., Bicking, J.B., Kwong, S.F., and Jones, J.H. (1967) J. Med. Chem. 10, 66-75. Benos, D.J. (1982) Am. J. Physiol. 242:C131-C145. Kleyman, T.R., Cragoe, E.J. Jr. (1988) J. Membrane Biol. 105, 1-21. Simchowitz, L., Cragoe, E.J., Jr. (1986) Mol. Pharmnacol. 30, 112-120. Simchowitz, L., Woltersdorf, O.W., Cragoe, E.J. Jr. (1987) J. Biol. Chem. 262, 1587515975.
687