(Received for publication, April 18, 1995; and in revised form, June 14, 1995)
From the
The function of the putative amphipathic helices between residues 236 and 314 of CTP:phosphocholine cytidylyltransferase was examined by constructing two truncation mutants; CT314 was missing the entire phosphorylation segment, whereas CT236 was missing both the region with the putative amphipathic helices and the phosphorylation segment. Stable cells lines expressing these truncation mutants in Chinese hamster ovary 58 cells were isolated and characterized. CT314 was predominantly soluble in control cells but became membrane-associated in cells treated with oleate, which also causes translocation of wild-type cytidylyltransferase. CT236 was found to be soluble both in control cells and in cells treated to cause translocation. These results strongly suggest that the membrane-binding site is located within residues 237-314. When assayed for activity in vitro, the mutant forms were catalytically active in the presence of exogenous lipids. CT236, moreover, was as active in the absence of lipids as in their presence, whereas CT314 required lipids for activity. The rate of phosphatidylcholine synthesis in cells expressing CT236 was considerably higher than in wild-type cells, consistent with the enzyme being constitutively active in the cells. These results indicate that residues 237-314 constitute an inhibitory segment; when this segment is removed from the catalytic domain by truncation or by binding to membranes, an inhibitory constraint is removed and cytidylyltransferase is activated.
The major pathway for phosphatidylcholine biosynthesis in
mammalian cells is the CDP choline pathway. The formation of the
activated intermediate, CDP choline, is catalyzed by the second enzyme
of the pathway, CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15),
which is a key regulatory enzyme for this pathway(1) . When
cultured cells are subjected to choline starvation (2, 3) or various treatments that stimulate
phosphatidylcholine
synthesis(4, 5, 6, 7) ,
cytidylyltransferase changes from a relatively inactive, soluble form
to an active, membrane-associated form. The soluble form is
intranuclear, and the membrane form is associated with the nuclear
envelope(8, 9, 10) . In some circumstances
active cytidylyltransferase appears to be associated with a lipoprotein
complex rather than a membrane(11, 12, 13) .
When assayed in vitro, the soluble form requires lipids for
activity, whereas the membrane form is usually quite active in the
absence of exogenous lipids. Thus it appears that the soluble form is
an inactive reservoir available to be activated when an increased
requirement for phosphatidylcholine synthesis is sensed. The soluble
form is highly phosphorylated and translocation of cytidylyltransferase
to membranes is accompanied by extensive
dephosphorylation(14, 15, 16, 17) .
The phosphorylation state of cytidylyltransferase, however, does not
appear to be a critical determinant of the distribution of the enzyme. ()
Sequences have been determined for several mammalian
cytidylyltransferase cDNAs (18, 19, 20, 21) and the gene from Saccharomyces cerevisiae(22) . The mammalian protein
has a molecular weight of about 42,000 and appears to be a
dimer(13, 23) . A comparison of the mammalian and
yeast sequences reveals a region from residues 79-209 that is
highly conserved and is proposed to be the catalytic
domain(21) . That this region is catalytic is supported by the
similarity of this region to a small bacterial CTP:glycerol-3-phosphate
cytidylyltransferase that is only as big as the putative catalytic
domain of the eukaryotic cytidylyltransferases (24, 25, 26) . The phosphorylation domain
extends from residue 315 to the COOH terminus(17) , and the
nuclear localization sequence is near the NH terminus (27) (see Fig.1).
Figure 1: Regions of cytidylyltransferase.
Between the phosphorylation region
and the catalytic domain is a sequence of about 80 residues that is
predicted to form amphipathic -helices(21) . Cornell and
co-workers proposed that this amphipathic helical region is a
membrane-binding domain for cytidylyltransferase (21) . In
support of this hypothesis, Craig et al., (28) showed
by chymotryptic digestion that there is a correlation between the
presence of the amphipathic helical region in a proteolytic fragment of
cytidylyltransferase and the ability of the fragment to bind to lipids in vitro. The digested enzyme was not reported to be active,
however, so it was not clear if the protease caused such a major
structural change in the enzyme that a membrane-binding site distinct
from the helical region might have been altered. Furthermore, it is not
clear if binding to lipids in vitro occurs by the same
mechanisms as binding to the nuclear envelope in vivo.
In the present manuscript we have expressed and characterized truncated forms of cytidylyltransferase that are missing either the phosphorylation region (CT314, Fig.1) or both the phosphorylation region and the amphipathic helical region (CT236). The truncated enzymes were examined for activity in vitro, subcellular location, ability to translocate, and ability to complement the phenotypes of a cell line in which the endogenous cytidylyltransferase is temperature-sensitive.
For the pulse-chase analysis, cells in 60-mm
dishes were washed with CMF-PBS twice and then incubated with 1.5 ml of
Ham's F-12 medium containing 5% fetal bovine serum plus 5
µCi/ml [H]choline for 30 min at 34 °C.
The labeled medium was then replaced with Ham's F-12 medium plus
5% fetal bovine serum and incubated at 40 °C for the indicated
time. Cells were harvested as described above for continuous labeling.
The lipid fraction was dried and counted to determine label in
choline-containing lipids. The aqueous phase was dried under air
overnight, and choline metabolites were separated on silica gel 60A
thin layer plates as described(33) .
Figure 2: Mobility and phosphorylation of wild-type cytidylyltransferase and truncation mutants. Cells were labeled at 34 °C, immunoprecipitated, electrophoresed, and blotted as described under ``Experimental Procedures.'' A, Western blot; B, autoradiogram of the blot. Lanes 1, wild-type cytidylyltransferase; lanes 2, CT314; lanes 3, CT236.
Because the extensive truncations of cytidylyltransferase might affect the catalytic activity of the enzyme, it was important to determine if the truncated forms were as active as the wild-type enzyme. Enzymatic activity in crude extracts was measured in the presence of activating lipids, and the amount of cytidylyltransferase protein was quantitated from Western blots (Table1). In the presence of the activating lipids, each mutant was quite active; there was no significant difference in mutant and wild-type activity.
Western blots of soluble and particulate fractions confirmed that the distribution of cytidylyltransferase protein followed that of cytidylyltransferase activity (Fig.3). Identical distributions in control and oleate-treated cells were observed by Western blotting for two different clonal isolates of each truncation mutant, indicating that the distributions in Table2were a property of the truncated enzyme and not an artifact of the specific clone (data not shown).
Figure 3: Subcellular distribution of cytidylyltransferase protein. Cells were treated in the absence (A) or the presence (B) of oleate at 40 °C and harvested as described in Table1. Identical volumes of the cell extracts were subjected to electrophoresis. This experiment was performed twice with the clonal isolate used here and once with a different clonal isolate for each mutant. Similar results were also obtained with a transient transfection. From the left the lanes correspond to wild-type soluble, wild-type particulate, CT314 soluble, CT314 particulate, CT236 soluble, and CT236 particulate.
Figure 4:
Incorporation of
[H]choline into cells expressing wild-type and
mutant cytidylyltransferases. A-E, pulse-chase protocol.
Cells were labeled with 5 µCi/ml [
H]choline
for 30 min at 34 °C and then incubated with unlabeled medium at 40
°C for the indicated time.
, CHO 58;
, wild type;
, CT314;
, CT236. A, phosphocholine; B,
CDP choline; C, phosphatidylcholine; D,
glycerophosphocholine; E, the ratio of CDP choline to
phosphocholine. F, a continuous labeling experiment. Cells
were labeled with 2 µCi/ml [
H]choline for at
40 °C for the indicated times. The amount of label incorporated
into phosphatidylcholine is shown. The pulse-chase experiment was
performed once; the continuous incorporation experiment was performed
four times with similar results.
Figure 5:
Cell growth at 40 °C. Stable cell
lines expressing wild-type or mutant cytidylyltransferases were plated
at 2 10
and incubated at 34 °C for 1 day and
then shifted to 40 °C for the indicated time. Viable cells were
determined by trypan blue exclusion. Two dishes were counted each day.
Symbols are as in Fig.4.
These studies have characterized two truncation mutants of cytidylyltransferase in which the enzyme was shortened by either 53 or 131 residues, a reduction in length of 14 or 36%, respectively. The catalytic activities of these truncation mutants were quite similar to that of wild-type cytidylyltransferase, however. This supports the concept that the catalytic region is truly a structurally distinct domain that can exist by itself as a catalytically active unit. The fact that these mutants were active also indicated that the truncations did not cause a global structural alteration in enzyme structure and allowed us to use the truncation mutants to investigate the functions of the regions that were deleted.
When the truncation mutants were
expressed in stable cell lines, their subcellular distribution was
altered compared with that of the wild type. The extent of membrane
association was higher for mutant CT314 (about 20%) than for wild-type
cytidylyltransferase (about 5%), consistent with the concept that the
dephosphorylated enzyme has a higher affinity for membranes. This
behavior has been previously observed with a cytidylyltransferase
mutant in which all 16 phosphorylation sites were mutated from Ser to
Ala. Like the 16S
A mutant, (
)a high
proportion CT314 was soluble even in control cells, further indicating
that dephosphorylation per se does not trigger membrane
binding.
The most striking feature of the subcellular location of the truncation mutants was the fact that CT236 was soluble, even in cells treated to cause the enzyme to translocate. The fact that this enzyme remains soluble but active provides very strong support for the hypothesis that the putative amphipathic helical structure that is predicted to be formed by residues 236-315 constitutes the membrane-binding domain of cytidylyltransferase(18) . It might be argued that the amphipathic helical region only affects membrane binding indirectly, for instance by influencing the region amino-terminal of the catalytic domain to form the actual membrane-binding domain. The fact that a synthetic peptide corresponding to residues 236-293 can bind to lipid vesicles that activate cytidylyltransferase but not those that do not activate, however, supports the hypothesis that the amphipathic helical region itself is truly the membrane-binding domain(37) . The involvement of amphipathic helices in membrane binding is a relatively new concept that is strongly supported by the crystal structure of prostaglandin H synthase, in which amphipathic helices are proposed to mediate membrane binding(38) .
Studies on the effect of lipids on the activities of the truncation mutants revealed that CT236 is not activated by lipids. In fact, CT236 is apparently constitutively active both in the cell as well as in enzymatic assays. Not only is the rate of phosphatidylcholine synthesis increased in cells expressing CT236, but the rate of production of glycerophosphocholine is also increased, a phenomenon that occurs in cells expressing high levels of cytidylyltransferase activity(39) . Thus the membrane-binding region serves as an autoinhibitory region. This would be consistent with the amphipathic helices interacting closely with the catalytic domain in order to inhibit activity. When the membrane-binding domain interacts with a suitably activating membrane surface, the helices would move away from the catalytic domain and inhibition would be released.
It is important to note that membrane binding does not
necessarily trigger activation, however. For example, the amount of
membrane-associated CT314 is considerably higher than wild type, yet
the rate of phosphatidylcholine synthesis in cells expressing CT314 is
the same as in cells expressing wild-type cytidylyltransferase.
Likewise, the level of membrane-bound cytidylyltransferase with the
16SA mutations is about 10 times higher than wild type, yet the
rate of phosphatidylcholine synthesis in the 16S
A cells is less
than 2-fold higher than wild type.
This suggests that an
additional factor is necessary for full activation of
membrane-associated cytidylyltransferase. Whether that factor is lipid
or protein in nature remains to be determined.
Cytidylyltransferase undergoes changes in its phosphorylation state during the cell cycle; these changes correlate with altered rates of phosphatidylcholine synthesis(40) . It is therefore surprising that CT236 and CT314 are fully able to complement the growth phenotype of CHO 58 cells. The membrane-binding and phosphorylation segments, therefore, do not appear to be necessary for progression through the CHO cell cycle under standard culture conditions. It is difficult to believe that a third of the polypeptide chain of cytidylyltransferase is useless to the cells, however. Like the unexpected location of cytidylyltransferase in the nucleus, the seeming dispensability of the membrane-binding and phosphorylation segments presents an enigma that remains to be addressed in the future.