(Received for publication, September 6, 1995; and in revised form, October 10, 1995)
From the
Cytochrome b is an amphipathic mobile
membrane protein that is predominantly located at the endoplasmic
reticulum surface. It is an essential component of a number of
membrane-bound redox systems. In animal and fungal cells cytochrome b
is thought to be an electron donor for sterol
modifying enzymes and fatty acid desaturases. Disruption of the
Saccharomyces cytochrome b
gene, however, yielded
cells that had no nutritional requirement for either sterols or
unsaturated fatty acids. Expression of sterol and fatty acid-modifying
genes was increased in the cytochrome b
-disrupted
cells, however, suggesting that cytochrome b
may
play some nonessential role in these functions. Unsaturated fatty acids
in yeast are formed by Ole1p, an oxygen-dependent
-9 fatty acid
desaturase that is an intrinsic endoplasmic reticulum membrane protein.
Although the yeast
-9 fatty acid desaturase does not appear to
require cytochrome b
, introduction of the rat
liver stearoyl-CoA desaturase gene into an ole1-disrupted,
cytochrome b
-disrupted yeast strain revealed that
this enzyme specifically requires cytochrome b
to
function. Comparison of the coding sequences of the yeast and rat
desaturase genes showed that the yeast protein contains a 113-amino
acid carboxyl-terminal extension not found in the rat enzyme. That
extension has regions of strong homology to cytochrome b
, particularly in the heme binding and electron
transfer motifs. Truncation or disruption of the desaturase cytochrome b
-like domain in cells that contain the wild type
diffusible b
produced unsaturated fatty acid
auxotrophy, suggesting that the cytochrome b
-like
domain of Ole1p plays an essential role in the desaturase reaction.
Cytochrome b is a ubiquitous eukaryotic
protein that appears to be an essential component of a number of
endoplasmic reticulum-linked redox enzyme systems. Its function has
been indicated in the modification of xenobiotic substances by
cytochromes P450 (1) and lipogenic enzyme systems that include
fatty acid desaturation(2, 3) , sterol biosynthesis (4) and fatty acid elongation(5) . Its ability to
function in such diverse enzymatic reactions is apparently due to its
structure, in which the large hydrophilic catalytic domain is linked to
a short carboxyl-terminal hydrophobic peptide sequence that serves as a
membrane anchor. This allows the heme protein to diffuse laterally
across the membrane surface and orient its heme moiety with its
electron donor and acceptors in a manner that allows for rapid electron
transfer. A recent study of cytochrome b
suggests
that it has a number of highly dynamic surfaces that may, in part,
allow it to interact with many different substrates(6) .
Evidence for the requirement of a heme-containing electron donor in
lipogenic systems comes from the requirement for sterols and
unsaturated fatty acids in heme-deficient yeast mutants(7) .
However, Truan et al.(8) indicated that disruption of
cytochrome b does not produce a nutritional
requirement for sterols. In this paper we show that disrupted
cytochrome b
does not affect the production of
unsaturated fatty acids. This, and the observation that heme-deficient
yeast mutants require both sterols and unsaturated fatty acids,
suggests that a minor cytochrome b
isoform, or an
alternative heme-containing electron donor plays a role in these
essential reactions.
In the yeast Saccharomyces, the OLE1 gene encodes the microsomal -9 fatty acid
desaturase(9) . In animal and fungal cells monounsaturated
fatty acids are aerobically synthesized from saturated fatty acids by
this intrinsic, membrane-bound desaturase. A double bond is inserted
between the 9- and 10-carbons of palmitoyl (16:0) and stearoyl (18:0)
CoA to form palmitoleic (16:1) and oleic (18:1) acids. In the proposed
reaction mechanism electrons are transferred from NADH-dependent
cytochrome b
reductase, via the heme-containing
cytochrome b
molecule, to the
-9 fatty acid
desaturase(3) . It had previously been shown that the rat liver
desaturase, driven by the OLE1 promoter and containing the
first 27 amino acids of Ole1p, will rescue an OLE1-disrupted
strain(9) . Cells containing this chimeric gene were observed
to have equivalent growth rates and only modest fatty acid
compositional changes to wild type cells. This further indicated that
the rat liver desaturase efficiently interacts with the yeast redox
system. The apparent nonrequirement for cytochrome b
in fatty acid desaturation and sterol
biosynthesis in gene-disrupted strains of yeast led us to examine the
role of cytochrome b
in the expression and
function of yeast endoplasmic reticulum lipogenic systems. This paper
indicates that the Saccharomyces fatty acid desaturase is a
modular protein that contains a carboxyl-terminal cytochrome b
-like domain. Similar domains are absent from the
cytochrome b
-dependent rat liver desaturase and
from yeast cytochrome P450 sterol biosynthetic enzymes. However,
cytochrome b
appears to play some role in the
regulation of these endoplasmic reticulum-based electron transport
systems, as disruption of the cytochrome b
gene
causes significant changes in the level of gene expression in the
cytochrome P450-like ERG11 gene and in the fatty acid
desaturase gene.
A gene disruption fragment was
produced by removing a 282-base pair HindIII fragment that
included both promoter and 5` protein coding sequences from the 1.4-kb
YSCYb5 gene. A HindIII-linkered KasI-HpaI LEU2 fragment from YEP351 (14) was inserted within the
region created by the HindIII digestion. An XbaI-XhoI fragment was used for the linear
transformation of DTY-10a and -11a, generating yeast strains AMY-1a and
AMY-1 ( Fig. 1and Table 1).
Figure 1:
PCR
strategy for cloning and disruption of the yeast cytochrome b.
Figure 2: Structure of the 4.8-kb chromosomal OLE1 gene.
From
earlier studies of heme biosynthetic mutants (7) it is known
that a heme-containing electron donor is required for the formation of
ergosterol and unsaturated fatty acids in Saccharomyces. It
was therefore expected that if cytochrome b was
required for fatty acid desaturation and sterol biosynthesis,
disruption of cytochrome b
would either knockout
or adversely affect the level of these components within the yeast
cell. Both Southern and Northern analysis ( Fig. 3and 4) were
performed to confirm that the disrupting DNA fragment replaced the
authentic cytochrome b
gene in the transformed
cells. The Southern blot of EcoRI-cut genomic DNA prepared
from DTY-10a and AMY-1a (Table 1) was probed with a
[
-
P]dATP-labeled 1.4-kb cytochrome b
DNA fragment (Fig. 3). This analysis
confirmed that there was a single copy of the cytochrome b
gene in DTY-10a and that this had been disrupted
with the LEU2 marker in AMY-1a. The LEU2 gene
contains a EcoRI site and therefore gives two bands on the
Southern blot.
Figure 3:
Southern blot analysis of cytochrome b-disrupted yeast. Genomic DNA isolated from wild
type strain DTY10a and strain AMY-1a were cut with EcoRI. The
resulting blots were probed with the 1.4-kb DNA fragment that
encompasses the cytochrome b
gene. A unique EcoRI site within the LEU2 gene gives rise to two
fragments of the predicted molecular size for insertion within the
cytochrome b
locus.
Figure 4:
Northern blot analysis of wild type
(DTY10a) and two independent cytochrome b disruptants (AMY-1a and -1
). Left panel, phosphor
images of RNA blots probed with radiolabeled OLE1 and
cytochrome b
DNA fragments. Right panel,
blots probed with radiolabeled DNA fragments from HMG1 (encoding HMG CoA reductase, a sterol biosynthetic enzyme), ERG11 (a cytochrome P450 sterol demethylase), and ribosomal
protein L32A.
Previous enzyme reconstitution
experiments by Strittmatter et al.(3, 18) had indicated that mammalian fatty acid
desaturase enzymes have a specific requirement for cytochrome b as an electron donor. The Saccharomyces
9 fatty acid desaturase was initially thought to be similar
in function to its homologous mammalian enzyme, given a well
established heme requirement for the formation of unsaturated fatty
acids(19, 20) . Based on those observations,
disruption of cytochrome b
in a haploid yeast
strain should cause the cells to become unsaturated fatty acid
auxotrophs. The prototrophy for unsaturated fatty acids in the
cytochrome b
-disrupted strain, however, suggested
that the microsomal cytochrome b
is not essential
for fatty acid desaturation. The observations that heme-deficient
mutants of yeast are auxotrophic for unsaturated fatty acids indicate
that the desaturase reaction requires a heme-containing molecule. In
light of these findings there must be a previously unidentified
alternative electron donor to the desaturase.
Figure 5:
Multiple copy plasmids containing either
the intact OLE1 gene (YEp OLE1) or the rat stearoyl
CoA-desaturase coding sequences fused to the OLE1 promoter and
its first 27 amino acids (YEp rat -9) were transformed into an OLE1-disrupted strain, AMY-2a (top half) and OLE1, cytochrome b
-disrupted strain
AMY-5
(bottom half). Cells were streaked on fatty
acid-free synthetic complete agar containing 2% glucose as the carbon
source. Plates were incubated at 30 °C for 4
days.
Figure 6:
Pileup
diagram of complete protein coding sequences of the yeast desaturase,
Ole1p, the rat stearoyl-CoA desaturase, and the yeast cytochrome b. Shaded regions in line groups
3, 4, and 6 of the comparison show the location
of conserved histidine motifs associated with the diiron-oxo moieties
in the desaturase regions of the yeast and rat proteins. Yeast
cytochrome b
aligns only with the
carboxyl-terminal region of Ole1p starting at residue 413. Shaded
regions in line group 8 indicate the position of
conserved amino acids that correspond to a heme-binding
pocket.
Figure 7:
Pileup diagram of animal, plant, and Saccharomyces cytochrome b protein coding
sequences compared with the Ole1p cytochrome b
-like domain (OLE1.pep). Shaded regions indicate the position of conserved amino acids that
correspond to a heme-binding pocket common to cytochrome b
peptides.
Figure 8:
Three-dimensional diagram of bovine
cytochrome b5 redrawn from Mathews et al.(23) showing positions of conserved amino acids with the
cytochrome b-like domain of Ole1p, antiparallel
helices associated with the heme crevice, and
-pleated sheet
structures that form the floor of the heme
pocket.
A significant difference between the cytochrome b domain of Ole1p and other cytochrome b
proteins is the number of acidic residues located in the region
that corresponds to the bovine cytochrome b
residues 36-83. This area consists of the heme-binding
pocket and residues that appear to be involved in the protein
donor/acceptor interaction sites for a number of cytochrome b
-mediated electron transfer reactions. Mammalian
cytochrome b
proteins have 12 acidic residues
within this region. By comparison plant cytochrome b
s have 13, the autonomous Saccharomyces cytochrome b
has 11, and Ole1p has only 5
corresponding acidic residues. In diffusible cytochrome b
proteins, the surface residues in this region
appear to be part of a highly dynamic face of the protein that must
adapt its conformation to interact with numerous
substrates(6) . By contrast, the Ole1p cytochrome b
-like domain presumably has only one
electron-accepting substrate and therefore may not need to form as many
charge-pair interactions to dock with the desaturase domain.
To create a chromosomal
disruption within the cytochrome b domain of OLE1, the vector carrying the BsmFI-PacI
deletion was recut at the BsmFI site (which was not destroyed)
to allow insertion of the LEU2 gene. A linear DNA fragment of
this construct was used to transform the wild type DTY-10a strain.
Expression of this gene in the resulting transformants (AMY-4
)
theoretically produces a 458-amino acid polypeptide consisting of the
amino-terminal 410-residue ``desaturase domain'' of OLE1, the NH
-terminal 8 residues of the cytochrome b
-like domain, and an additional 50 residues
derived from bases in the upstream promoter region of LEU2.
This disruption of the native OLE1 gene also produced
transformants that were auxotrophic for unsaturated fatty acids in
cells that also contain a functional native cytochrome b
gene.
To determine if these modified strains were, in fact,
synthesizing the appropriate OLE1-encoding mRNAs, Northern
blot analysis was performed on cells containing the native OLE1 gene (DTY-11a), OLE1-disrupted cells (AMY-3)
containing plasmids bearing either the native OLE1 gene or the
cytochrome b
motif-disrupted form of OLE1 under the control of the GAL1 promoter, and cells
containing the chromosomal form of OLE1 with a disrupted
cytochrome b
motif (AMY-4
). We have
previously shown that unsaturated fatty acids repress OLE1 expression at the level of transcription and mRNA
stability(24, 25) . Because unsaturated acids are
required for growth of cells that contain the disrupted forms of OLE1, cells were first grown to a density of 2
10
/ml in the presence of 0.5 mM 16:1 and 0.5
mM 18:1 (in the repressed state). The cells were then washed
and incubated in fatty acid-free medium to induce expression of the
modified genes. Phosphor image analysis of the blots using the Saccharomyces PGK1 gene as an internal control indicated that
under these conditions the mRNA from the wild type OLE1 gene
was induced 18-fold following transfer to fatty acid-free medium.
Messenger RNA from the chromosomal truncated OLE1 gene was
induced from undetectable levels in the repressed state to
approximately that seen with the wild type gene under derepressed
conditions (Fig. 9). That mRNA was at a significantly lower
molecular weight than the wild type. Messenger RNAs produced under
control of the GAL1 promoter that encoded either the native OLE1 protein coding sequence or the cytochrome b
motif-disrupted form of the protein were
expressed in both unsaturated fatty acid-fed and washed cells. (
)In derepressed cells, those mRNA levels were,
respectively, 81 and 45% of the wild type species. We were unable to
determine if the proteins produced by these constructs are expressed.
Attempts to detect native Ole1p using antibodies generated against the
NH
-terminal and COOH-terminal ends of the protein have not
been successful. (
)
Figure 9:
Northern blot of wild type (DTY-10a),
AMY-3 + YCpGAL-OLE, AMY-3
+ YCpGAL-OLE
b
, and AMY-4
total RNA under
repressed (unsaturated fatty acid-supplemented) and derepressed (no
fatty acid supplement) conditions. Cultures were grown to a density of
2
10
cells/ml in synthetic galactose medium and
unsaturated fatty acids as described under ``Results.'' At
times 0 aliquots were taken for mRNA isolation. The remaining cells
were washed and resuspended in nonsupplemented synthetic galactose
medium and grown for 2 h to derepress transcription of genes under
control of the OLE1 promoter prior to isolation of total RNA.
Blots of the fractionated RNAs were probed with radiolabeled OLE1 and PGK1 gene fragments.
Given that deletion of residues within
this domain blocks the function of the yeast desaturase, these
experiments suggest that the cytochrome b-like
domain functions as a primary electron donor to the desaturase domain
of Ole1p. We cannot rule out the possibility, however, that the
residual carboxyl-terminal peptide sequences in the truncated forms of
Ole1p may block the ability of the diffusible cytochrome b
to participate in electron transfer that
normally occurs in wild type cells. With certain substrates, cytochrome b
is known to provide a second electron to
cytochrome P450 mixed function oxidases (26) . The
approximately 2-fold increase in OLE1 mRNA levels in
cytochrome b
gene-disrupted cells may represent a
compensation mechanism for decreased catalytic efficiency in the
absence of the diffusible cytochrome b
. Neither
can we rule out the possibility that a truncated form of Ole1p similar
to that of the homologous rat desaturase (with an appropriate
combination of COOH-terminal residues) can accept electrons from the
diffusible membrane-bound cytochrome b
.
The
chimeric yeast desaturase gene appears to have evolved through an event
in which the NH-terminal protein coding regions of an
ancestral cytochrome b
gene were fused to the
carboxyl terminus of coding sequences within an independent desaturase
gene. There may be some selective advantage for this type of enzyme
system, given that cytochrome b
in liver fatty
acid desaturation appears to diffuse laterally across the membrane
surface among NADH cytochrome b
reductase, its
electron donor, and the desaturase(27, 28) . Tethering
the cytochrome b
to the desaturase could
potentially speed up the electron transfer by presenting a correctly
oriented heme group with respect to the dioxo-iron cluster, eliminating
the need for diffusion and reorientation of the reduced cytochrome b
. The linkage of the heme domain with the
desaturase domain of Ole1p raises questions, however, concerning the
docking sites for electron transfer among cytochrome b
, its electron donor, and electron acceptor.
Fusion of the cytochrome b
domain to the
desaturase probably results in a relatively fixed orientation of heme
group with respect to the dioxo-iron center. If the membrane-bound form
of NADH reductase or a similar membrane-tethered reductase acts as the
electron donor to the Ole1p heme group, the docking site of the
electron donor may reside in a different location than the site of
electron transfer between the heme moiety and the desaturase iron
cluster.