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Insights from inducible membranes
ROBIN WRIGHT
MEMBRANEBIOGENESIS
Insights from indu ible membranes .’ The analysis of membrane arrays induced in yeast by over-expressing certain membrane proteins is providing new insights into the general problem of membrane biogenesis and morphology.
It is nearly impossible these days to pick up a biological science journal that does not report new forays into the realm of protein targeting. This is hardly surprising, as one of the great success stories of modern cell biology is our current understanding of how cells put proteins in their proper places. We seem, in fact, to be close to having a description of protein targeting at molecular resolution. Unfortunately, the sophistication of our knowledge about protein targeting mechanisms contrasts sharply witlh our basic ignorance of a related issue - how do cells regulate the synthesis and
Fig. 1. Three-dimensional nuclear yellow
870
reconstruction envelope and ER membranes. cap on one side of the nucleus
from optical sections Karmellae membranes, (see [8]).
0 Current
organization of the specific membranes into or through which proteins are inserted? The analysis of inducible membranes in yeast is providing some insight into this question. . As in other nucleated cells, the endoplasmic reticulum (ER) of yeast cells can undergo dramatic alterations in amount and organization. Some of these alterations can be induced merely by raising the levels of a subset of ER membrane proteins. One of the best characterized of these is 3-hydroxyl-3-methylglutaryl coenzyme A
of a living yeast cell stained induced by over-expressing
Biology
with DiOC,, a lipophilic dye that accumulates the protein HMG-CoA reductase, are visible
1993, Vol 3 No 12
in as a
DISPATCH
Fig, 2. The
HMG-CoA
reductas’e
Cytochrome
bS
Cytochrome
P450
predicted topologies of karmella-inducing proteins. Although these three proteins can all induce karmellae, their only common structural features are their localization in the ER and the disposition of their catalytic domains on the cytosolic side of the bilayer.
“ER lumen
reductase (HMG-COA reductase). In mammalian cells 111,and in the yeasts Saccharomyces cereznkiae [2,31 and Schizosaccharomyces po;mbe (RW and PY Lum, unpublished observations), inc:reases in HMG-CoA reductase levels lead to the cell-type specific proliferation of ER membranes. In mammalian cells, HMG-CoA reductaseinduced membranes begin as outfoldings of the outer nuclear envelope that subsequently differentiate into hexagonally packed arrays of smooth ER tubules, known as crystalloid Eli 141. Yeast cells, by contrast, assemble stacks of flattened membrane cisternae in the cytosol or in close association with the nucleus. The nucleus-associated memlbrane stacks have been called karmellae (Fig. 1). Thus, the experimental increase in levels of proteins such as HMG-CoA reductase can serve of a molecular switch to turn on specific membrane synthesis, providing a convenient system for investigating the mechanisms by which cells regulate membrane biogenesis and morphology. To understand how certain proteins can induce changes in membrane assembly, it is necessary to consider their enzymatic activities. HMG-CoA reductase catalyzes the rate-limiting step in sterol biosynthesis 151,so it is conceivable that the effects on membranes of increasing HMG-CoA reductase levels simply reflect changes in cellular sterol content. However, the formation of neither crystalloid ER nor karmellae requires the enzymatic activity of HMG-CoA reductase (161 and RW, unpublished observations). It turns out that the ability of HMG-CoA reductase to induce changes in ER membrane assembly resides in its membrane domain, which has no direct enzymatic role in sterol metabolism (Fig. 2). An alternative possibility is that the ability of HMG-CoA reductase to induce membrane changes reflects homologous interactions between the membrane domains of HMG-CoA reductase molecules. However, we have isolated mutant yeast strains that do not assemble karmellae even though their HMG-CoA reductase levels are high (S Nygaard and RW, unpublished observations). Thus, increased HMG-CoA reductase levels alone are no’t sufficient to induce karmella formation and addihonal gene products must be required, In order to ‘understand how cells assemble karmellae, therefore,. we must decipher both how protein membrane domains deliver a ‘signal’ to the cell and how that signal is ‘transduced’. Recent evidence
that karmella formation can be induced by at least three other proteins sheds light on both these issues. Several groups have reported that membrane arrays that are morphologically identical to karmellae are induced in S. cereznkiae in response to the expression of certain ER-localized cytochromes: the Candida maltosa cytochrome P4s0enzymes, P-450Cml and P-450Cm2 171, and rat cytochrome b, 181. Interestingly, both cytochrome b, and cytochrome P4s0are ER-localized, hemebinding proteins with potential roles in the metabolism of sterols and lipids. The cytochrome P4s0proteins comprise a large family of enzymes that catalyze the attachment of molecular oxygen (generally as a hydroxyl group) to a variety of endogenous substrates, including sterols, and exogenous compounds, including lipophilic drugs. This reaction requires the activity of an ERlocalized electron transport chain, of which cytochrome P 450 forms a part. The specificity of the reaction depends on the particular cytochrome P,,,: for example, the C. maltosa P-450Cml and P-450Cm2 enzymes are involved in the metabolism of alkanes as a carbon source; other cytochrome P4s0 enzymes in the same cells catalyze late steps in sterol metabolism. Cytochrome b, is required for the desaturation and elongation of fatty acids and sterol biosynthesis, and can also function in cytochrome P,,+M-mediated reactions. Thus, whereas HMG-CoA reductase is involved in the earliest step of sterol biosynthesis, both types of cytochrome have roles in late steps of the sterol biosynthetic pathway and in lipid metabolism. As the effect of HMG-CoA reductase on membrane proliferation is mediated by its membrane domain rather than its catalytic domain, it is reasonable to predict that the membrane domains of all the karmella-inducing proteins might have similar sequences. However, the membrane domains of HMG-CoA reductase, cytochrome P4s0 and cytochrome b, lack any obvious amino-acid sequence similarity, and even have different membrane-crossing topologies (Fig. 2). Unlike the complicated membrane domain of HMG-CoA reductase, the membrane domains of cytochrome I’,~, and cytochrome b, are relatively simple, being composed of one or two transmembrane regions (the issue of whether cytochrome b, actually passes through the membrane, or is just tethered by a hairpin loop that
871
872
Current Biology 1993,
‘Vol
3 No
12
Fig. 3. Analysis of the karmelia-inducing ability of cytochrome b, mutant proteins (data from 191).
only partially enters the bilayer, remains controversial, but for simplicity I have assumed that the former is true). Also, cytochrjome b, is not targeted to the ER membrane by a typical signal sequence, like HMG-CoA reductase and cytochrome Pb5*, but instead is targeted post-translationally to the ER. Thus, the only apparent common features of the three known types of protein that can induce karmellae are their localization in the ER and their roles in sterol metabolism. Vergeres and coworkers 181have begun to examine the features of cytochrome b, that are important for its ability to induce membrane changes. They placed the rat cytochrome b, gene under the control of the S. cerevisiae CUP2 promoter, which is activated by growth of the yeast cells in the presence of copper ions. When cytochrome b, expression was induced by the addition of copper ions, karmellae were assembled in a proportion of the cells in the population (they are never assembled in 100% of the cells, in part, at least, because karmellae are asymmetrically segregated at mitosis). Vergeres et al. 181 demonstrated that the relative level of karmella assembly was directly proportional to the amount of cytochrome b, produced: karmellae were present in more cells and with more layers per cell when cytochrome b, expression levels were higher. Like HMG-CoA reductase, the association of cytochrome b, with ER membranes, but not its catalytic activity, is essential for karmella induction. Thus, the soluble, catalytic: carboxy-terminal domain did not induce membrane alterations, showing that simply increasing the cytochrome b, activity level is not sufficient for karmella induction. Vergeres et al. [8] also examined whether the catalytic activity of cytochrome b, was necessary for membrane assembly. This analysis relied on the inhibition of heme formation by treatment of cells with Syc:cinyl acetone, When cytochrome b, expression was:,i,nduced in the inhibited cells, hemedeficient, catalytically inactive apocytochrome b, accumulated and normal karmella membranes were assembled. Taken together, these experiments demonstrate that increases in the cytochrome b, catalytic
activity level are neither necessary nor sufficient for membrane alterations. Instead, the presence of the protein within ER membranes appears to be the critical factor for triggering membrane biogenesis. These results suggest that the signal for karmella formation might be present within the membrane domain itself of the inducing proteins. To test this possibility, several forms of cytochrome b, with altered membrane domains were examined (Fig. 3). Because the proline at position 115 in the predicted transmembrane region figures prominently in models of membrane topology, Vergeres et al. 181changed that proline to an alanine, and found that the mutant protein induced normal karmella membranes with similar efficiency to the wildtype protein. In contrast, changing the alanine at position 116 to a proline substantially reduced the protein’s ability to induce karmella membranes, though the karmellae that were induced had a normal morphology. The Ala 116 Pro mutation creates a proline pair in the center of the putative transmembrane region of cytochrome b,, and it is possible that the mutant protein’s decreased ability to induce karmellae results from the proline pair altering the topology of the transmembrane region. Alternatively, the mutation might remove a residue in the transmembrane region that is critical for generation of the signal that induces karmella formation. To distinguish between these possibilities, the 22 amino acids of the transmembrane region were completely replaced with 22 leucine residues. The polyleucine sequence should form a very hydrophobic a-helical domain, but it lacks any sequence similarity tc the normal transmembrane region. Surprisingly although the proportion of cells with karmellae waz reduced, and the resulting membrane stacks had 5 slightly different organization to those induced by the wild-type protein, the polyleucine mutant is capable 01 inducing karmella formation. This argues that tht sequences required for karmella induction lie outside the transmembrane region. If so, the role of the trans membrane region in karmella formation may simply bt to deliver the protein to the prop,er membrane environ ment, or to maintain it in that location after targeting.
DISPATCH
Experiments such as th,ose of Vergeres et al. [81 are beginning to map the spbecific features, or ‘signals’, that enable certain membrane proteins to exert profound effects on membrane assembly and organization. This information is an essential step toward the goal of understanding how cells modulate the assembly of specific membiines. However, many questions will remain even when these studies have been completed. Are the karmellae induced by cytochrome b, identical to those induced by HMG-CoA reductase? Do the karmellainducing proteins define a pathway for karmella induction, in which one protein induces another in the pathway, or does each Iindependently induce karmella assembly? Does the ability to induce karmellae reflect specific functions of a sub-domain of the ER? Why cannot all ER membrane proteins produce alterations in ER organization? The answer to these questions will require elucidation of the mechanisms by which the signals delivered by the karmella-inducing proteins are transduced. As with many complicated questions in ceil biology, perhaps the most efficient approach is a genetic one that enables the yeast cell itself to point the way. References 1.
CHIN DJ, LUSKEY KL, ~DERSON RGW, FAUST JR, GOLDSTEIN JL, BROWN MS: Appearance of crystalloidendoplasmicreticulum
in compact&resistant
Chinese hamster cells with a 500-fold
elevation in 3-hydroxy-3-methylglutarylcoenzyme A reductase. Proc Nat1 Acad Sci USA 1992, 79:1185-1189.
R, Bassos M, D’ARI L, RINE J: Increased amounts of HMG-CoA reductase induce “karmellae”: a proliferation of stacked membrane pairs surrounding the yeast nucleus. J Cell Biol1988, 107:101-114. 3. WRIGHT R, KELLER G, GOULD SJ, SUBRAMANI S, Rrh% J: Cell-type control of membrane biogenesis induced by HMG-CoA reductase overproduction. New Biologist 1930, 2915-921. 4. PATHAK RK, LUSKEY L, ANDERSON RGW: Biogenesis of the crystalloid endoplasmic reticulum in UT-l cells: evidence that newly formed endoplasmic reticulum emerges from the nuclear envelope. JCell Biol1986, 102:2158-2168. 5. GOLDSTEIN JL, BROWN MS: Regulation of the mevalonate pathway. Nature 1990, 343:425-430. 6. GIL G, FAUST JR, CHIN DJ, GOLDSTEIN JL, BROWN MS: Membrane-bound domain of HMG-CoA reductase is required for sterol-enhanced degradation of the enzyme. Cell 1985,
2.
WRIGHT
7.
SCHUNCK W, VOCEL B, GROSS E, KARGEL S, MAUERSBERGER K, KOPKE K, GENGNAGEL C, MULLER H: Comparison of two
41:249-258.
cytochromes P-450 from Candida maltosa: primary structures, substratespecficities and effects of their expression in Saccharomyces cerevtsiae on the proliferation of the endoplasmic reticulum. EurJCell Bioll991, 553336345. 8. VERGERES G, YEN TSB, AGGEL.ER J, LAUSIER J, WASKELL L: A model system for studying membrane biogenesis: overexpression of cytochrome b, in yeast results in marked proliferation of the intracellular membrane. J Cell Sci 1993, 106:249-259. 9. KONING A, LUM PY, WILLIAMS J, WRIGHT R: DiOC, staining reveals organelle structure and dynamics in living yeast cells. CellMotil Cytoskell993, 25:111- 128.
Robin Wright, Department of Zoology, University Washington, Seattle, Washington 98195, USA.
of
873
MEMBRANEBIOGENESIS
Insights from indu ible membranes .’ The analysis of membrane arrays induced in yeast by over-expressing certain membrane proteins is providing new insights into the general problem of membrane biogenesis and morphology.
It is nearly impossible these days to pick up a biological science journal that does not report new forays into the realm of protein targeting. This is hardly surprising, as one of the great success stories of modern cell biology is our current understanding of how cells put proteins in their proper places. We seem, in fact, to be close to having a description of protein targeting at molecular resolution. Unfortunately, the sophistication of our knowledge about protein targeting mechanisms contrasts sharply witlh our basic ignorance of a related issue - how do cells regulate the synthesis and
Fig. 1. Three-dimensional nuclear yellow
870
reconstruction envelope and ER membranes. cap on one side of the nucleus
from optical sections Karmellae membranes, (see [8]).
0 Current
organization of the specific membranes into or through which proteins are inserted? The analysis of inducible membranes in yeast is providing some insight into this question. . As in other nucleated cells, the endoplasmic reticulum (ER) of yeast cells can undergo dramatic alterations in amount and organization. Some of these alterations can be induced merely by raising the levels of a subset of ER membrane proteins. One of the best characterized of these is 3-hydroxyl-3-methylglutaryl coenzyme A
of a living yeast cell stained induced by over-expressing
Biology
with DiOC,, a lipophilic dye that accumulates the protein HMG-CoA reductase, are visible
1993, Vol 3 No 12
in as a
DISPATCH
Fig, 2. The
HMG-CoA
reductas’e
Cytochrome
bS
Cytochrome
P450
predicted topologies of karmella-inducing proteins. Although these three proteins can all induce karmellae, their only common structural features are their localization in the ER and the disposition of their catalytic domains on the cytosolic side of the bilayer.
“ER lumen
reductase (HMG-COA reductase). In mammalian cells 111,and in the yeasts Saccharomyces cereznkiae [2,31 and Schizosaccharomyces po;mbe (RW and PY Lum, unpublished observations), inc:reases in HMG-CoA reductase levels lead to the cell-type specific proliferation of ER membranes. In mammalian cells, HMG-CoA reductaseinduced membranes begin as outfoldings of the outer nuclear envelope that subsequently differentiate into hexagonally packed arrays of smooth ER tubules, known as crystalloid Eli 141. Yeast cells, by contrast, assemble stacks of flattened membrane cisternae in the cytosol or in close association with the nucleus. The nucleus-associated memlbrane stacks have been called karmellae (Fig. 1). Thus, the experimental increase in levels of proteins such as HMG-CoA reductase can serve of a molecular switch to turn on specific membrane synthesis, providing a convenient system for investigating the mechanisms by which cells regulate membrane biogenesis and morphology. To understand how certain proteins can induce changes in membrane assembly, it is necessary to consider their enzymatic activities. HMG-CoA reductase catalyzes the rate-limiting step in sterol biosynthesis 151,so it is conceivable that the effects on membranes of increasing HMG-CoA reductase levels simply reflect changes in cellular sterol content. However, the formation of neither crystalloid ER nor karmellae requires the enzymatic activity of HMG-CoA reductase (161 and RW, unpublished observations). It turns out that the ability of HMG-CoA reductase to induce changes in ER membrane assembly resides in its membrane domain, which has no direct enzymatic role in sterol metabolism (Fig. 2). An alternative possibility is that the ability of HMG-CoA reductase to induce membrane changes reflects homologous interactions between the membrane domains of HMG-CoA reductase molecules. However, we have isolated mutant yeast strains that do not assemble karmellae even though their HMG-CoA reductase levels are high (S Nygaard and RW, unpublished observations). Thus, increased HMG-CoA reductase levels alone are no’t sufficient to induce karmella formation and addihonal gene products must be required, In order to ‘understand how cells assemble karmellae, therefore,. we must decipher both how protein membrane domains deliver a ‘signal’ to the cell and how that signal is ‘transduced’. Recent evidence
that karmella formation can be induced by at least three other proteins sheds light on both these issues. Several groups have reported that membrane arrays that are morphologically identical to karmellae are induced in S. cereznkiae in response to the expression of certain ER-localized cytochromes: the Candida maltosa cytochrome P4s0enzymes, P-450Cml and P-450Cm2 171, and rat cytochrome b, 181. Interestingly, both cytochrome b, and cytochrome P4s0are ER-localized, hemebinding proteins with potential roles in the metabolism of sterols and lipids. The cytochrome P4s0proteins comprise a large family of enzymes that catalyze the attachment of molecular oxygen (generally as a hydroxyl group) to a variety of endogenous substrates, including sterols, and exogenous compounds, including lipophilic drugs. This reaction requires the activity of an ERlocalized electron transport chain, of which cytochrome P 450 forms a part. The specificity of the reaction depends on the particular cytochrome P,,,: for example, the C. maltosa P-450Cml and P-450Cm2 enzymes are involved in the metabolism of alkanes as a carbon source; other cytochrome P4s0 enzymes in the same cells catalyze late steps in sterol metabolism. Cytochrome b, is required for the desaturation and elongation of fatty acids and sterol biosynthesis, and can also function in cytochrome P,,+M-mediated reactions. Thus, whereas HMG-CoA reductase is involved in the earliest step of sterol biosynthesis, both types of cytochrome have roles in late steps of the sterol biosynthetic pathway and in lipid metabolism. As the effect of HMG-CoA reductase on membrane proliferation is mediated by its membrane domain rather than its catalytic domain, it is reasonable to predict that the membrane domains of all the karmella-inducing proteins might have similar sequences. However, the membrane domains of HMG-CoA reductase, cytochrome P4s0 and cytochrome b, lack any obvious amino-acid sequence similarity, and even have different membrane-crossing topologies (Fig. 2). Unlike the complicated membrane domain of HMG-CoA reductase, the membrane domains of cytochrome I’,~, and cytochrome b, are relatively simple, being composed of one or two transmembrane regions (the issue of whether cytochrome b, actually passes through the membrane, or is just tethered by a hairpin loop that
871
872
Current Biology 1993,
‘Vol
3 No
12
Fig. 3. Analysis of the karmelia-inducing ability of cytochrome b, mutant proteins (data from 191).
only partially enters the bilayer, remains controversial, but for simplicity I have assumed that the former is true). Also, cytochrjome b, is not targeted to the ER membrane by a typical signal sequence, like HMG-CoA reductase and cytochrome Pb5*, but instead is targeted post-translationally to the ER. Thus, the only apparent common features of the three known types of protein that can induce karmellae are their localization in the ER and their roles in sterol metabolism. Vergeres and coworkers 181have begun to examine the features of cytochrome b, that are important for its ability to induce membrane changes. They placed the rat cytochrome b, gene under the control of the S. cerevisiae CUP2 promoter, which is activated by growth of the yeast cells in the presence of copper ions. When cytochrome b, expression was induced by the addition of copper ions, karmellae were assembled in a proportion of the cells in the population (they are never assembled in 100% of the cells, in part, at least, because karmellae are asymmetrically segregated at mitosis). Vergeres et al. 181 demonstrated that the relative level of karmella assembly was directly proportional to the amount of cytochrome b, produced: karmellae were present in more cells and with more layers per cell when cytochrome b, expression levels were higher. Like HMG-CoA reductase, the association of cytochrome b, with ER membranes, but not its catalytic activity, is essential for karmella induction. Thus, the soluble, catalytic: carboxy-terminal domain did not induce membrane alterations, showing that simply increasing the cytochrome b, activity level is not sufficient for karmella induction. Vergeres et al. [8] also examined whether the catalytic activity of cytochrome b, was necessary for membrane assembly. This analysis relied on the inhibition of heme formation by treatment of cells with Syc:cinyl acetone, When cytochrome b, expression was:,i,nduced in the inhibited cells, hemedeficient, catalytically inactive apocytochrome b, accumulated and normal karmella membranes were assembled. Taken together, these experiments demonstrate that increases in the cytochrome b, catalytic
activity level are neither necessary nor sufficient for membrane alterations. Instead, the presence of the protein within ER membranes appears to be the critical factor for triggering membrane biogenesis. These results suggest that the signal for karmella formation might be present within the membrane domain itself of the inducing proteins. To test this possibility, several forms of cytochrome b, with altered membrane domains were examined (Fig. 3). Because the proline at position 115 in the predicted transmembrane region figures prominently in models of membrane topology, Vergeres et al. 181changed that proline to an alanine, and found that the mutant protein induced normal karmella membranes with similar efficiency to the wildtype protein. In contrast, changing the alanine at position 116 to a proline substantially reduced the protein’s ability to induce karmella membranes, though the karmellae that were induced had a normal morphology. The Ala 116 Pro mutation creates a proline pair in the center of the putative transmembrane region of cytochrome b,, and it is possible that the mutant protein’s decreased ability to induce karmellae results from the proline pair altering the topology of the transmembrane region. Alternatively, the mutation might remove a residue in the transmembrane region that is critical for generation of the signal that induces karmella formation. To distinguish between these possibilities, the 22 amino acids of the transmembrane region were completely replaced with 22 leucine residues. The polyleucine sequence should form a very hydrophobic a-helical domain, but it lacks any sequence similarity tc the normal transmembrane region. Surprisingly although the proportion of cells with karmellae waz reduced, and the resulting membrane stacks had 5 slightly different organization to those induced by the wild-type protein, the polyleucine mutant is capable 01 inducing karmella formation. This argues that tht sequences required for karmella induction lie outside the transmembrane region. If so, the role of the trans membrane region in karmella formation may simply bt to deliver the protein to the prop,er membrane environ ment, or to maintain it in that location after targeting.
DISPATCH
Experiments such as th,ose of Vergeres et al. [81 are beginning to map the spbecific features, or ‘signals’, that enable certain membrane proteins to exert profound effects on membrane assembly and organization. This information is an essential step toward the goal of understanding how cells modulate the assembly of specific membiines. However, many questions will remain even when these studies have been completed. Are the karmellae induced by cytochrome b, identical to those induced by HMG-CoA reductase? Do the karmellainducing proteins define a pathway for karmella induction, in which one protein induces another in the pathway, or does each Iindependently induce karmella assembly? Does the ability to induce karmellae reflect specific functions of a sub-domain of the ER? Why cannot all ER membrane proteins produce alterations in ER organization? The answer to these questions will require elucidation of the mechanisms by which the signals delivered by the karmella-inducing proteins are transduced. As with many complicated questions in ceil biology, perhaps the most efficient approach is a genetic one that enables the yeast cell itself to point the way. References 1.
CHIN DJ, LUSKEY KL, ~DERSON RGW, FAUST JR, GOLDSTEIN JL, BROWN MS: Appearance of crystalloidendoplasmicreticulum
in compact&resistant
Chinese hamster cells with a 500-fold
elevation in 3-hydroxy-3-methylglutarylcoenzyme A reductase. Proc Nat1 Acad Sci USA 1992, 79:1185-1189.
R, Bassos M, D’ARI L, RINE J: Increased amounts of HMG-CoA reductase induce “karmellae”: a proliferation of stacked membrane pairs surrounding the yeast nucleus. J Cell Biol1988, 107:101-114. 3. WRIGHT R, KELLER G, GOULD SJ, SUBRAMANI S, Rrh% J: Cell-type control of membrane biogenesis induced by HMG-CoA reductase overproduction. New Biologist 1930, 2915-921. 4. PATHAK RK, LUSKEY L, ANDERSON RGW: Biogenesis of the crystalloid endoplasmic reticulum in UT-l cells: evidence that newly formed endoplasmic reticulum emerges from the nuclear envelope. JCell Biol1986, 102:2158-2168. 5. GOLDSTEIN JL, BROWN MS: Regulation of the mevalonate pathway. Nature 1990, 343:425-430. 6. GIL G, FAUST JR, CHIN DJ, GOLDSTEIN JL, BROWN MS: Membrane-bound domain of HMG-CoA reductase is required for sterol-enhanced degradation of the enzyme. Cell 1985,
2.
WRIGHT
7.
SCHUNCK W, VOCEL B, GROSS E, KARGEL S, MAUERSBERGER K, KOPKE K, GENGNAGEL C, MULLER H: Comparison of two
41:249-258.
cytochromes P-450 from Candida maltosa: primary structures, substratespecficities and effects of their expression in Saccharomyces cerevtsiae on the proliferation of the endoplasmic reticulum. EurJCell Bioll991, 553336345. 8. VERGERES G, YEN TSB, AGGEL.ER J, LAUSIER J, WASKELL L: A model system for studying membrane biogenesis: overexpression of cytochrome b, in yeast results in marked proliferation of the intracellular membrane. J Cell Sci 1993, 106:249-259. 9. KONING A, LUM PY, WILLIAMS J, WRIGHT R: DiOC, staining reveals organelle structure and dynamics in living yeast cells. CellMotil Cytoskell993, 25:111- 128.
Robin Wright, Department of Zoology, University Washington, Seattle, Washington 98195, USA.
of
873