Planta
Planta 150, 58-69 (1980)
9 by Springer-Verlag 1980
The Isolation of Endoplasmic Reticulum from Barley Aleurone Layers Russell L. Jones Department of Botany, University of California, Berkeley, CA 94720, USA
Abstract. Techniques for the isolation and purification of endoplasmic reticulum (ER) from aleurone layers of barley ( H o r d e u m vulgare L.) were assessed. Neither differential centrifugation nor density gradient centrifugation of a homogenate separate the ER or other organelles of this tissue from the lipidcontaining spherosomes. Isopycnic sucrose gradient centrifugation of organelles first purified by molecular sieve chromatography on Sepharose 4B, however, results in separation of the organelles based on their differing buoyant densities. Manipulation of the magnesium concentration of the isolation media and density-gradient solutions affords isolation of ER at a density of 1.13-1.14g cc 1 and 1.17-1.18g cc -1. Electron microscopy shows that the membranes sedimenting at 1.13-1.14 g cc-1 are devoid of ribosomes and are characteristic of smooth ER, while those sedimenting at 1.17 1.18 g cc -1 are studded with ribosomes and have the features of rough ER. Endoplasmic reticulum isolated by isopycnic density gradient centrifugation can be further purified by rate-zonal centrifugation. Key words: A l e u r o n e - a-Amylase Endoplasmic reticulum - H o r d e u m - Organelle isolation.
Introduction Electron-microscope (Jones 1969; Jones and Price 1970; Paleg and Hyde 1964; Vigil and Ruddat 1973) and biochemical (Evins and Varner 1971; Johnson and Kende 1971 ; Koehler and Varner 1973) evidence indicated that the endoplasmic reticulum (ER) of barAbbreviations." E D T A - ethylenediaminetetraacetic acid; ER= endoplasmic reticulum; GA gibberellin; GA3=gibberellic acid; Trizma = tris(hydroxymethyl)aminomethane
0032-0935/80/0150/0058/$02.4o
ley aleurone layers underwent dramatic changes following treatment with the hormone gibberellic acid (GA3). These changes were thought to involve an increase in the amount of ER cisternae as well as an increase in the stacking of these membrane lamellae. Based on these observations it was hypothesized that the changes in barley ER were important in both the synthesis and the secretion of the GA-induced hydrolases (Johnson and Kende 1971; Varner and Ho 1976). The organization of ER into complex stacks is a characteristic of secretory cells in animal tissue (for review, see Fawcett 1966), and it has been demonstrated that the polyribosomes associated with the ER membranes are involved in the synthesis of the secreted protein (Redman et al. 1966). Recent evidence also implicates the ER membrane directly in the post-translational modification of secretory proteins, resulting in their vectorial transport across the ER into the lumen of the cisternum (Blobel and Dobberstein 1975; see review by Wickner 1979). The view that GA affects the synthesis or organization of ER in cereal aleurone has been challenged. Firn and Kende (1974) in a study of lipid metabolism in barley, and Varty and Laidman (1976) studying wheat aleurone were unable to demonstrate an increase in the predominant classes of phospholipids after incubation in GA3 and concluded that there was neither a qualitative nor a quantitative effect of GA on phospholipid metabolism in these tissues. Using electron microscopy to study wheat aleurone Colborne et al. (1976) were unable to demonstrate an increase over controls in the ER of aleurone cells of whole grains imbided in GA3 for up to 6 d or of endosperm imbibed in GA3 for 4 d. The role of the ER in the transport of hydrolases from the aleurone layer has also been debated. The evidence from electron microscopy is equivocal. Thus, while Jones (1969) and Vigil and Ruddat (1973) suggested that ER- or dictyosome-derived vesicles might
R.L. Jones: Isolation of ER from Barley Aleurone
be involved in hydrolase secretion, their evidence was circumstantial. Cell-fractionation studies have also failed to provide rigorous evidence for the pathway of enzyme secretion from aleurone cells. I (Jones 1972) proposed that since 95% of the a-amylase present in an aleurone cell homogenate was not associated with a particulate fraction, the release of the enzyme might occur by direct passage of enzyme molecules across the plasma membrane. In contrast, Gibson and Paleg (1976) proposed that the secreted hydrolases of wheat were transported by lysosomes, while Firn (1975) and Locy and Kende (1978) proposed that ER-derived vesicles were involved in this process. To resolve the conflicting views concerning the nature and function of the ER of the cereal aleurone, I have developed techniques for the quantitative isolation, identification and manipulation of the ER of barley aleurone. In this paper, various methods for the isolation of barley aleurone ER are evaluated and the smooth and rough membranes isolated are described. In a subsequent paper (Jones 1980) changes in this membrane system during imbibition and incubation and its role in the transport of hydrolases will be reported.
Material and Methods Plant Material. Grains (caryopses) of barley (Hordeum vulgare L. cv. Himalaya; 1974 harvest; A g r o n o m y Department, University of Washington, Pulhnan, Wash., USA) were de-embryonated, and the embryo-less "half-seeds" were imbibed on sterile sand moistened with H 2 0 (Jones and Varner i967) or on sterile filter paper moistened with 20 m M succinate buffer (pH 5.3) and 10 m M CaC12 (modified from Firn and K e n d e 1974) for 3 d at 25 ~ C. Aleurone layers were removed from the imbibed ihalf-seeds under steriie conditions and incubated at 25 ~ C in the presence or absence of 50 lam GA3 in either sterile 2 m M acetate buffer (pH 4.87) containing 10 m M CaC12 (layers from H20-imbibed half-seeds; Jones and Varner 1967) or sterile 20 m M succinate buffer (pH 5.32) plus 10 m M CaC12 (layers from buffer-imbibed half-seeds; Firn and Kende 1974). Since imbibition and incubation conditions have no bearing on the results of the organelle isolation procedures described in this paper, they are not discussed here but are considered in detail in the subsequent paper (Jones 1980). Homogenization. Aleurone layers were homogenized at 2 ~ C with a motorized razor blade chopper in either low-Mg 2 + or high-Mg 2 + buffer, modified from Breidenbach et al. (1968) and Locy and Kende (1978). Low-Mg 2+ homogenization buffer contained 50 m M tris(hydroxymethyl)aminomethane (Trizma), pH 7.4 (Sigma Chemical Co., St. Louis, MO., USA), 0.56 M (18.5%, w/w) sucrose (grade I; Sigma), 10 m M KC1, 0.1 m M MgC12 and 2 m M ethylenediamenetetraacetic acid (EDTA, tetrasodium salt; Sigma). H i g h - M g 2+ homogenization buffer contained 0.56 M sucrose, 5 0 m M Trizma, p H 7 . 4 , 1 0 m M K C 1 , 3 m M M g C 1 2 and l m M EDTA. The homogenate was filtered through two layers of bleached cheesecloth with two buffer rinses of 0.5 ml each, and the cell debris was discarded. AI1 subsequent procedures were carried out at 2M ~ C. The filtered homogenate was centrifuged at 1080.g for 10 min (Sorvall SS34 rotor; D u P o n t Instruments, New-
59 Filtered homogenote [
l
[lO00.g
I
Dilferential
centrifugation
Gel filtration
Planta 150, 58-69 (1980)
9 by Springer-Verlag 1980
The Isolation of Endoplasmic Reticulum from Barley Aleurone Layers Russell L. Jones Department of Botany, University of California, Berkeley, CA 94720, USA
Abstract. Techniques for the isolation and purification of endoplasmic reticulum (ER) from aleurone layers of barley ( H o r d e u m vulgare L.) were assessed. Neither differential centrifugation nor density gradient centrifugation of a homogenate separate the ER or other organelles of this tissue from the lipidcontaining spherosomes. Isopycnic sucrose gradient centrifugation of organelles first purified by molecular sieve chromatography on Sepharose 4B, however, results in separation of the organelles based on their differing buoyant densities. Manipulation of the magnesium concentration of the isolation media and density-gradient solutions affords isolation of ER at a density of 1.13-1.14g cc 1 and 1.17-1.18g cc -1. Electron microscopy shows that the membranes sedimenting at 1.13-1.14 g cc-1 are devoid of ribosomes and are characteristic of smooth ER, while those sedimenting at 1.17 1.18 g cc -1 are studded with ribosomes and have the features of rough ER. Endoplasmic reticulum isolated by isopycnic density gradient centrifugation can be further purified by rate-zonal centrifugation. Key words: A l e u r o n e - a-Amylase Endoplasmic reticulum - H o r d e u m - Organelle isolation.
Introduction Electron-microscope (Jones 1969; Jones and Price 1970; Paleg and Hyde 1964; Vigil and Ruddat 1973) and biochemical (Evins and Varner 1971; Johnson and Kende 1971 ; Koehler and Varner 1973) evidence indicated that the endoplasmic reticulum (ER) of barAbbreviations." E D T A - ethylenediaminetetraacetic acid; ER= endoplasmic reticulum; GA gibberellin; GA3=gibberellic acid; Trizma = tris(hydroxymethyl)aminomethane
0032-0935/80/0150/0058/$02.4o
ley aleurone layers underwent dramatic changes following treatment with the hormone gibberellic acid (GA3). These changes were thought to involve an increase in the amount of ER cisternae as well as an increase in the stacking of these membrane lamellae. Based on these observations it was hypothesized that the changes in barley ER were important in both the synthesis and the secretion of the GA-induced hydrolases (Johnson and Kende 1971; Varner and Ho 1976). The organization of ER into complex stacks is a characteristic of secretory cells in animal tissue (for review, see Fawcett 1966), and it has been demonstrated that the polyribosomes associated with the ER membranes are involved in the synthesis of the secreted protein (Redman et al. 1966). Recent evidence also implicates the ER membrane directly in the post-translational modification of secretory proteins, resulting in their vectorial transport across the ER into the lumen of the cisternum (Blobel and Dobberstein 1975; see review by Wickner 1979). The view that GA affects the synthesis or organization of ER in cereal aleurone has been challenged. Firn and Kende (1974) in a study of lipid metabolism in barley, and Varty and Laidman (1976) studying wheat aleurone were unable to demonstrate an increase in the predominant classes of phospholipids after incubation in GA3 and concluded that there was neither a qualitative nor a quantitative effect of GA on phospholipid metabolism in these tissues. Using electron microscopy to study wheat aleurone Colborne et al. (1976) were unable to demonstrate an increase over controls in the ER of aleurone cells of whole grains imbided in GA3 for up to 6 d or of endosperm imbibed in GA3 for 4 d. The role of the ER in the transport of hydrolases from the aleurone layer has also been debated. The evidence from electron microscopy is equivocal. Thus, while Jones (1969) and Vigil and Ruddat (1973) suggested that ER- or dictyosome-derived vesicles might
R.L. Jones: Isolation of ER from Barley Aleurone
be involved in hydrolase secretion, their evidence was circumstantial. Cell-fractionation studies have also failed to provide rigorous evidence for the pathway of enzyme secretion from aleurone cells. I (Jones 1972) proposed that since 95% of the a-amylase present in an aleurone cell homogenate was not associated with a particulate fraction, the release of the enzyme might occur by direct passage of enzyme molecules across the plasma membrane. In contrast, Gibson and Paleg (1976) proposed that the secreted hydrolases of wheat were transported by lysosomes, while Firn (1975) and Locy and Kende (1978) proposed that ER-derived vesicles were involved in this process. To resolve the conflicting views concerning the nature and function of the ER of the cereal aleurone, I have developed techniques for the quantitative isolation, identification and manipulation of the ER of barley aleurone. In this paper, various methods for the isolation of barley aleurone ER are evaluated and the smooth and rough membranes isolated are described. In a subsequent paper (Jones 1980) changes in this membrane system during imbibition and incubation and its role in the transport of hydrolases will be reported.
Material and Methods Plant Material. Grains (caryopses) of barley (Hordeum vulgare L. cv. Himalaya; 1974 harvest; A g r o n o m y Department, University of Washington, Pulhnan, Wash., USA) were de-embryonated, and the embryo-less "half-seeds" were imbibed on sterile sand moistened with H 2 0 (Jones and Varner i967) or on sterile filter paper moistened with 20 m M succinate buffer (pH 5.3) and 10 m M CaC12 (modified from Firn and K e n d e 1974) for 3 d at 25 ~ C. Aleurone layers were removed from the imbibed ihalf-seeds under steriie conditions and incubated at 25 ~ C in the presence or absence of 50 lam GA3 in either sterile 2 m M acetate buffer (pH 4.87) containing 10 m M CaC12 (layers from H20-imbibed half-seeds; Jones and Varner 1967) or sterile 20 m M succinate buffer (pH 5.32) plus 10 m M CaC12 (layers from buffer-imbibed half-seeds; Firn and Kende 1974). Since imbibition and incubation conditions have no bearing on the results of the organelle isolation procedures described in this paper, they are not discussed here but are considered in detail in the subsequent paper (Jones 1980). Homogenization. Aleurone layers were homogenized at 2 ~ C with a motorized razor blade chopper in either low-Mg 2 + or high-Mg 2 + buffer, modified from Breidenbach et al. (1968) and Locy and Kende (1978). Low-Mg 2+ homogenization buffer contained 50 m M tris(hydroxymethyl)aminomethane (Trizma), pH 7.4 (Sigma Chemical Co., St. Louis, MO., USA), 0.56 M (18.5%, w/w) sucrose (grade I; Sigma), 10 m M KC1, 0.1 m M MgC12 and 2 m M ethylenediamenetetraacetic acid (EDTA, tetrasodium salt; Sigma). H i g h - M g 2+ homogenization buffer contained 0.56 M sucrose, 5 0 m M Trizma, p H 7 . 4 , 1 0 m M K C 1 , 3 m M M g C 1 2 and l m M EDTA. The homogenate was filtered through two layers of bleached cheesecloth with two buffer rinses of 0.5 ml each, and the cell debris was discarded. AI1 subsequent procedures were carried out at 2M ~ C. The filtered homogenate was centrifuged at 1080.g for 10 min (Sorvall SS34 rotor; D u P o n t Instruments, New-
59 Filtered homogenote [
l
[lO00.g
I
Dilferential
centrifugation
Gel filtration
