- Email: [email protected]
Soluble proteins, an often overlooked contaminant in plasma membrane preparations
Update
250
TRENDS in Plant Science
made over the past 30 years, and we have learned that an amazingly complex system of interactions for self-defense has evolved in plants. The recent work of the Howe group and the new questions raised are proof to this claim. Acknowledgements Work in my laboratory is partially supported by a grant from the University of South Carolina Research and Productive Scholarship Fund.
References 1 Green, T.R. and Ryan, C.A. (1972) Wound-induced proteinase inhibitor in plant leaves: a possible defense mechanism against insects. Science 175, 776 – 777 2 Ryan, C.A. (1990) Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annu. Rev. Phytopathol. 28, 425 – 449 3 Ryan, C.A. (2000) The systemin signaling pathway: differential activation of plant defensive genes. Biochim. Biophys. Acta 1477, 112 – 121 4 Lee, G.I. and Howe, G.A. (2003) The tomato mutant spr1 is defective in systemin perception and the production of a systemic wound signal for defense gene expression. Plant J. 33, 567 – 576 5 Li, L. et al. (2002) Distinct roles for jasmonate synthesis and action in the systemic wound response of tomato. Proc. Natl. Acad. Sci. U. S. A. 99, 6416 – 6421
Vol.8 No.6 June 2003
6 Orozco-Cardenas, M.L. et al. (1993) Expression of an antisense prosystemin gene in tomato plants reduces resistance toward Manduca sexta larvae. Proc. Natl. Acad. Sci. U. S. A. 90, 8273– 8276 7 Scheer, J. and Ryan, C.A. (2002) The systemin receptor SR160 from Lycopersicon peruvianum is a member of the LRR receptor kinase family. Proc. Natl. Acad. Sci. U. S. A. 99, 9585 – 9590 8 Howe, G.A. et al. (1996) An octadecanoid pathway mutant (JL5) of tomato is compromised in signaling for defense against insect attack. Plant Cell 8, 2067– 2077 9 Howe, G.A. and Ryan, C.A. (1999) Suppressors of systemin signaling identify genes in the tomato wound response pathway. Genetics 153, 1411 – 1421 10 Doares, S.H. et al. (1995) Oligogalacturonides and chitosan activate plant defensive genes through the octadecanoid pathway. Proc. Natl. Acad. Sci. U. S. A. 92, 4095 – 4098 11 Ryan, C.A. and Moura, D.S. (2002) Systemic wound signaling in plants: a new perception. Proc. Natl. Acad. Sci. U. S. A. 99, 6519 – 6520 12 Pearce, G. et al. (2001) Production of multiple plant hormones from a single polyprotein precursor. Nature 411, 817 – 820 13 Ryan, C.A. et al. (2002) Polypeptide hormones. Plant Cell 14, S251 – S264 1360-1385/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1360-1385(03)00106-7
| Letters
Soluble proteins, an often overlooked contaminant in plasma membrane preparations Alajos Be´rczi1 and Han Asard2 1 2
Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, POB 521, H-6701 Szeged, Hungary N 146, Beadle Center for Genetic Research, University of Nebraska – Lincoln, Lincoln, NE 68588-0664, USA
Identification of the subcellular localization of a particular protein provides essential information towards understanding its physiological function. Among other techniques, aqueous polymer two-phase partitioning (APTP) has become a widely used method to purify plant plasma membranes (PMs), and, consequently, to support claims for the association of a protein of interest with that membrane type. For example, in a recent paper [1], the involvement of a PM NADPH oxidase in abscisic acid- and water stress-induced antioxidant defense responses was suggested mainly on the basis of APTP. As is common practice, APTP-purified PM vesicles were prepared under iso-osmotic and low ionic strength conditions, and activity assays were performed using hypo-osmotic and low ionic strength buffers. However, it has been repeatedly shown that proteins can non-specifically associate to membranes under these conditions and therefore their unequivocal localization to the PM requires more thoughtful approaches. Here, we provide a critical analysis of membrane isolation conditions and suggestions to prevent premature conclusions. Since the introduction of APTP in the mid 1980s [2], we Corresponding author: Alajos Be´rczi ([email protected]). http://plants.trends.com
are now able to obtain chlorophyll-free PM fractions from green tissues [3] and to separate sealed inside-out and right-side-out PM vesicles [4]. APTP has also been shown to result in a purer population of PM vesicles, with respect to contamination by other membrane types, when compared to discontinuous sucrose density-gradient centrifugation [5,6]. However, the presence of other possible contaminants, soluble proteins in particular, is rarely addressed. An analysis of proteins identified in APTP-purified PM fractions from Arabidopsis thaliana by two-dimensional gel electrophoresis illustrates the extent of this contamination [7]. Fifty-one polypeptides out of the 82 spots sequenced and identified from the ‘PM’ fraction were at least as abundant in the supernatant as in the PM fraction [7], and some of these are soluble cytoplasmic proteins. This contamination probably originates from the tight sealing of PM vesicles upon disruption of the tissue. The lumen (interior) of these vesicles therefore might contain any molecules or small particles that are present in the homogenate. In addition to the soluble contaminants, and depending on the ionic strength of the homogenization buffer (HB), membrane vesicles can electrostatically bind charged
Update
TRENDS in Plant Science
compounds that might not be present on their surface in vivo. Thus, the composition of the HB, as well as the volume (or mass) ratio of HB to the tissue to be homogenized, will considerably affect the level of attached and/or trapped contaminants in the purified PM vesicles. Plant tissues are generally homogenized in a buffer containing 0.25–0.33 M sucrose. A sucrose-containing HB was introduced to mimic the osmotic condition of the cytosol to isolate intact mitochondria. However, this practice has never been re-evaluated with respect to the importance of other physico-chemical parameters such as ionic strength. The presence of soluble contaminants with the use of a low ionic strength, iso-osmotic HB, was realized early on in PM research, and a salt-washing step was introduced in several laboratories. PM vesicles were incubated in 0.5 M KCl or KI for 30 min and then collected by centrifugation before using them in any enzyme assays or protein purification steps (see e.g. [8,9]). Such a high-salt-washing step decreased the protein content of the PM fraction by , 30% [8]. A high-salt wash is appropriate to remove charged compounds loosely attached to the outer surface of membrane vesicles, but is unable to remove any possible contaminants from the vesicle interior. To remove these, the vesicles should be opened to release trapped soluble contaminants and non-specifically bound proteins from the inner surface. A thorough washing protocol to remove possible contaminants in PM fractions has been introduced [10,11] but is not widely used [1,7]. Using a thorough stripping protocol, which includes mild-detergent treatment and high-salt-washing steps, the protein content of the PM fraction decreases as much as 40% [10]. This level of contamination is therefore significant compared with the levels of the PM Hþ-pumping ATPase, which are estimated to be , 5% of the total PM proteins [12]. Thus, although APTP is a powerful method to obtain highly purified PM vesicles, soluble and non-specifically bound contaminants can be significant in these fractions, therefore conclusions about the localization of a PM can only be drawn when the correct homogenization and/or stripping conditions have been used.
Vol.8 No.6 June 2003
Acknowledgements This work was supported by Hungarian research grants of B-4/99 (OMFB) and T-034488 (OTKA). We are grateful to Julie Stone for valuable suggestions. A contribution of the University of Nebraska Agricultural Research Division, Lincoln, NE 68583. Journal Series No. 14078.
References 1 Jiang, M. and Zhang, J. (2002) Involvement of plasma-membrane NADPH oxidase in abscisic acid- and water stress-induced antioxidant defense in leaves of maize seedlings. Planta 215, 1022 – 1030 2 Larsson, C. (1983) Partition in aqueous polymer two-phase systems: a rapid method for separation of membrane particles according to their surface properties. In Isolation of Membranes and Organelles from Plant Cells (Hall, J.L. and Moore, A.L., eds) pp. 277– 309, Academic Press 3 Kjellbom, P. and Larsson, C. (1984) Preparation and polypeptide composition of chlorophyll-free plasma membranes from leaves of light-grown spinach and barley. Physiol. Plant. 62, 501 – 509 4 Larsson, C. et al. (1994) Isolation of highly purified plant plasma membranes and separation of inside-out and right-side-out vesicles. Methods Enzymol. 228, 451 – 469 5 Be´rczi, A. and Møller, I.M. (1986) Comparison of the properties of plasmalemma vesicles purified from wheat roots by phase partitioning and by discontinuous sucrose gradient centrifugation. Physiol. Plant. 68, 59 – 66 6 Hodges, T.K. and Mills, D. (1986) Isolation of the plasma membrane. Methods Enzymol. 118, 41 – 54 7 Santoni, V. et al. (1998) Use of a proteome strategy for tagging proteins present at the plasma membrane. Plant J. 16, 633 – 641 8 Briskin, D.P. and Poole, R.J. (1984) Characterization of the solubilized plasma membrane ATPase of red beet. Plant Physiol. 76, 26 – 30 9 Anthon, G.E. and Spanswick, R.M. (1986) Purification and properties of the Hþ-translocating ATPase from the plasma membrane of tomato roots. Plant Physiol. 81, 1080– 1085 10 Van Gestelen, P. et al. (1997) Solubilization and separation of a plant plasma membrane NADPH-O2 synthase from other NAD(P)H 2 oxidoreductases. Plant Physiol. 115, 543– 550 11 Be´rczi, A. and Møller, I.M. (1998) NADH-monodehydroascorbate oxidoreductase is one of the redox enzymes in spinach leaf plasma membranes. Plant Physiol. 116, 1029 – 1036 12 Serrano, R. (1985) ATPase and proton transport in isolated plasma membranes from plants and fungi. Plasma Membrane ATPase of Plants and Fungi, pp. 79 – 129, CRC Press 1360-1385/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1360-1385(03)00100-6
International Plant Photobiology Meeting 2–6 September 2003 Marburg, Germany This meeting covers the following topics: prokaryotic photoreceptors, phytochromes and phytochrome signalling, blue-light photoreceptors and blue-light signalling, light entrainment of the circadian clock, light, environment, and stress For more information contact: Alfred Batschauer Plant Physiology and Photobiology, Philipps-University Marburg, Karl-von-Frisch-Str. 8, 35032 Marburg, Germany e-mail: [email protected] https://cgi-host.uni-marburg.de/~ppmm2003/ http://plants.trends.com
251
250
TRENDS in Plant Science
made over the past 30 years, and we have learned that an amazingly complex system of interactions for self-defense has evolved in plants. The recent work of the Howe group and the new questions raised are proof to this claim. Acknowledgements Work in my laboratory is partially supported by a grant from the University of South Carolina Research and Productive Scholarship Fund.
References 1 Green, T.R. and Ryan, C.A. (1972) Wound-induced proteinase inhibitor in plant leaves: a possible defense mechanism against insects. Science 175, 776 – 777 2 Ryan, C.A. (1990) Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annu. Rev. Phytopathol. 28, 425 – 449 3 Ryan, C.A. (2000) The systemin signaling pathway: differential activation of plant defensive genes. Biochim. Biophys. Acta 1477, 112 – 121 4 Lee, G.I. and Howe, G.A. (2003) The tomato mutant spr1 is defective in systemin perception and the production of a systemic wound signal for defense gene expression. Plant J. 33, 567 – 576 5 Li, L. et al. (2002) Distinct roles for jasmonate synthesis and action in the systemic wound response of tomato. Proc. Natl. Acad. Sci. U. S. A. 99, 6416 – 6421
Vol.8 No.6 June 2003
6 Orozco-Cardenas, M.L. et al. (1993) Expression of an antisense prosystemin gene in tomato plants reduces resistance toward Manduca sexta larvae. Proc. Natl. Acad. Sci. U. S. A. 90, 8273– 8276 7 Scheer, J. and Ryan, C.A. (2002) The systemin receptor SR160 from Lycopersicon peruvianum is a member of the LRR receptor kinase family. Proc. Natl. Acad. Sci. U. S. A. 99, 9585 – 9590 8 Howe, G.A. et al. (1996) An octadecanoid pathway mutant (JL5) of tomato is compromised in signaling for defense against insect attack. Plant Cell 8, 2067– 2077 9 Howe, G.A. and Ryan, C.A. (1999) Suppressors of systemin signaling identify genes in the tomato wound response pathway. Genetics 153, 1411 – 1421 10 Doares, S.H. et al. (1995) Oligogalacturonides and chitosan activate plant defensive genes through the octadecanoid pathway. Proc. Natl. Acad. Sci. U. S. A. 92, 4095 – 4098 11 Ryan, C.A. and Moura, D.S. (2002) Systemic wound signaling in plants: a new perception. Proc. Natl. Acad. Sci. U. S. A. 99, 6519 – 6520 12 Pearce, G. et al. (2001) Production of multiple plant hormones from a single polyprotein precursor. Nature 411, 817 – 820 13 Ryan, C.A. et al. (2002) Polypeptide hormones. Plant Cell 14, S251 – S264 1360-1385/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1360-1385(03)00106-7
| Letters
Soluble proteins, an often overlooked contaminant in plasma membrane preparations Alajos Be´rczi1 and Han Asard2 1 2
Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, POB 521, H-6701 Szeged, Hungary N 146, Beadle Center for Genetic Research, University of Nebraska – Lincoln, Lincoln, NE 68588-0664, USA
Identification of the subcellular localization of a particular protein provides essential information towards understanding its physiological function. Among other techniques, aqueous polymer two-phase partitioning (APTP) has become a widely used method to purify plant plasma membranes (PMs), and, consequently, to support claims for the association of a protein of interest with that membrane type. For example, in a recent paper [1], the involvement of a PM NADPH oxidase in abscisic acid- and water stress-induced antioxidant defense responses was suggested mainly on the basis of APTP. As is common practice, APTP-purified PM vesicles were prepared under iso-osmotic and low ionic strength conditions, and activity assays were performed using hypo-osmotic and low ionic strength buffers. However, it has been repeatedly shown that proteins can non-specifically associate to membranes under these conditions and therefore their unequivocal localization to the PM requires more thoughtful approaches. Here, we provide a critical analysis of membrane isolation conditions and suggestions to prevent premature conclusions. Since the introduction of APTP in the mid 1980s [2], we Corresponding author: Alajos Be´rczi ([email protected]). http://plants.trends.com
are now able to obtain chlorophyll-free PM fractions from green tissues [3] and to separate sealed inside-out and right-side-out PM vesicles [4]. APTP has also been shown to result in a purer population of PM vesicles, with respect to contamination by other membrane types, when compared to discontinuous sucrose density-gradient centrifugation [5,6]. However, the presence of other possible contaminants, soluble proteins in particular, is rarely addressed. An analysis of proteins identified in APTP-purified PM fractions from Arabidopsis thaliana by two-dimensional gel electrophoresis illustrates the extent of this contamination [7]. Fifty-one polypeptides out of the 82 spots sequenced and identified from the ‘PM’ fraction were at least as abundant in the supernatant as in the PM fraction [7], and some of these are soluble cytoplasmic proteins. This contamination probably originates from the tight sealing of PM vesicles upon disruption of the tissue. The lumen (interior) of these vesicles therefore might contain any molecules or small particles that are present in the homogenate. In addition to the soluble contaminants, and depending on the ionic strength of the homogenization buffer (HB), membrane vesicles can electrostatically bind charged
Update
TRENDS in Plant Science
compounds that might not be present on their surface in vivo. Thus, the composition of the HB, as well as the volume (or mass) ratio of HB to the tissue to be homogenized, will considerably affect the level of attached and/or trapped contaminants in the purified PM vesicles. Plant tissues are generally homogenized in a buffer containing 0.25–0.33 M sucrose. A sucrose-containing HB was introduced to mimic the osmotic condition of the cytosol to isolate intact mitochondria. However, this practice has never been re-evaluated with respect to the importance of other physico-chemical parameters such as ionic strength. The presence of soluble contaminants with the use of a low ionic strength, iso-osmotic HB, was realized early on in PM research, and a salt-washing step was introduced in several laboratories. PM vesicles were incubated in 0.5 M KCl or KI for 30 min and then collected by centrifugation before using them in any enzyme assays or protein purification steps (see e.g. [8,9]). Such a high-salt-washing step decreased the protein content of the PM fraction by , 30% [8]. A high-salt wash is appropriate to remove charged compounds loosely attached to the outer surface of membrane vesicles, but is unable to remove any possible contaminants from the vesicle interior. To remove these, the vesicles should be opened to release trapped soluble contaminants and non-specifically bound proteins from the inner surface. A thorough washing protocol to remove possible contaminants in PM fractions has been introduced [10,11] but is not widely used [1,7]. Using a thorough stripping protocol, which includes mild-detergent treatment and high-salt-washing steps, the protein content of the PM fraction decreases as much as 40% [10]. This level of contamination is therefore significant compared with the levels of the PM Hþ-pumping ATPase, which are estimated to be , 5% of the total PM proteins [12]. Thus, although APTP is a powerful method to obtain highly purified PM vesicles, soluble and non-specifically bound contaminants can be significant in these fractions, therefore conclusions about the localization of a PM can only be drawn when the correct homogenization and/or stripping conditions have been used.
Vol.8 No.6 June 2003
Acknowledgements This work was supported by Hungarian research grants of B-4/99 (OMFB) and T-034488 (OTKA). We are grateful to Julie Stone for valuable suggestions. A contribution of the University of Nebraska Agricultural Research Division, Lincoln, NE 68583. Journal Series No. 14078.
References 1 Jiang, M. and Zhang, J. (2002) Involvement of plasma-membrane NADPH oxidase in abscisic acid- and water stress-induced antioxidant defense in leaves of maize seedlings. Planta 215, 1022 – 1030 2 Larsson, C. (1983) Partition in aqueous polymer two-phase systems: a rapid method for separation of membrane particles according to their surface properties. In Isolation of Membranes and Organelles from Plant Cells (Hall, J.L. and Moore, A.L., eds) pp. 277– 309, Academic Press 3 Kjellbom, P. and Larsson, C. (1984) Preparation and polypeptide composition of chlorophyll-free plasma membranes from leaves of light-grown spinach and barley. Physiol. Plant. 62, 501 – 509 4 Larsson, C. et al. (1994) Isolation of highly purified plant plasma membranes and separation of inside-out and right-side-out vesicles. Methods Enzymol. 228, 451 – 469 5 Be´rczi, A. and Møller, I.M. (1986) Comparison of the properties of plasmalemma vesicles purified from wheat roots by phase partitioning and by discontinuous sucrose gradient centrifugation. Physiol. Plant. 68, 59 – 66 6 Hodges, T.K. and Mills, D. (1986) Isolation of the plasma membrane. Methods Enzymol. 118, 41 – 54 7 Santoni, V. et al. (1998) Use of a proteome strategy for tagging proteins present at the plasma membrane. Plant J. 16, 633 – 641 8 Briskin, D.P. and Poole, R.J. (1984) Characterization of the solubilized plasma membrane ATPase of red beet. Plant Physiol. 76, 26 – 30 9 Anthon, G.E. and Spanswick, R.M. (1986) Purification and properties of the Hþ-translocating ATPase from the plasma membrane of tomato roots. Plant Physiol. 81, 1080– 1085 10 Van Gestelen, P. et al. (1997) Solubilization and separation of a plant plasma membrane NADPH-O2 synthase from other NAD(P)H 2 oxidoreductases. Plant Physiol. 115, 543– 550 11 Be´rczi, A. and Møller, I.M. (1998) NADH-monodehydroascorbate oxidoreductase is one of the redox enzymes in spinach leaf plasma membranes. Plant Physiol. 116, 1029 – 1036 12 Serrano, R. (1985) ATPase and proton transport in isolated plasma membranes from plants and fungi. Plasma Membrane ATPase of Plants and Fungi, pp. 79 – 129, CRC Press 1360-1385/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1360-1385(03)00100-6
International Plant Photobiology Meeting 2–6 September 2003 Marburg, Germany This meeting covers the following topics: prokaryotic photoreceptors, phytochromes and phytochrome signalling, blue-light photoreceptors and blue-light signalling, light entrainment of the circadian clock, light, environment, and stress For more information contact: Alfred Batschauer Plant Physiology and Photobiology, Philipps-University Marburg, Karl-von-Frisch-Str. 8, 35032 Marburg, Germany e-mail: [email protected] https://cgi-host.uni-marburg.de/~ppmm2003/ http://plants.trends.com
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