A dissection of Sec14p function in yeast has been driven by the analysis of suppressor mutations that endow cells with the ability to execute Golgi function, and retain viability, in the absence of Sec14p (Cleves et al., 1991a
; Kearns et al., 1998
). The logic on which suppressor genetics is founded dictates that such bypass Sec14p mutations exert their effects by restoring a biochemical condition that normally falls under the purview of Sec14p function. From these analyses, we have proposed that Sec14p functions to maintain a Golgi DAG pool that is critical for Golgi secretory function (McGee et al., 1994
; Kearns et al., 1997
). Specifically, we proposed that PtdCho-bound Sec14p down-regulates DAG consumption via the CDP-choline pathway (Skinner et al., 1995
; Alb et al., 1996
; Kearns et al., 1998
), whereas PtdIns-bound Sec14p promotes DAG generation by regulating inositol phospholipid metabolism (Fang et al., 1998
; Kearns et al., 1997
). In this manner, Sec14p serves as a phospholipid sensor whose phospholipid-bound states independently, but convergently, function to maintain Golgi DAG (Figure ).
Figure 8 Bypass Sec14p mechanisms. The relevant lipid metabolic pathways and proteins that determine the activity of these pathways are shown. The key lipids (i.e., PtdIns-4-P, PtdCho, and DAG) are indicated in bold. Key proteins and their corresponding sites (more ...)
One of several important lines of evidence supporting the DAG model was our observation that the ability of sac1
strains to effect bypass Sec14p correlated with what we interpreted as overproduction of an inositol phospholipid that we identified as the most highly modified yeast sphingolipid, M(IP)2
C, a lipid whose synthesis produces Golgi DAG (Kearns et al., 1997
). In those experiments, the failure of Kearns et al. (1997)
to use deacylation as an initial means for fractionating the deacylatable glycerophospholipids from sphingolipids contributed significantly to the misidentification. More rigorous analyses now indicate that the major inositol phospholipid that accumulates in sac1
strains is PtdIns-4-P (Figure ). A biochemical basis for elevations in PtdIns-4-P is suggested by the demonstration that the Sac1p domains of other inositide phosphatases themselves represent novel phosphoinositide phosphatase modules (Guo et al., 1999
). Thus, sac1
strains likely accumulate PtdIns-4-P because the primary mechanism for its degradation to PtdIns is inactivated.
Based on the collective data reported herein, we revise our interpretation of the mechanism for bypass Sec14p in sac1
strains to take into account the various new data (Figure ). We maintain that increased DAG production represents the key physiological event that allows Sec14p-independent growth and secretion in these strains, as previously proposed (Kearns et al., 1997
). The evidence now suggests that the pathway for this DAG production in sac1
mutants involves PLD activity. We also report the unanticipated discovery that the physiological basis for sac1
-associated inositol auxotrophy is related to aberrant lipid metabolism in these strains and not to defects in transcriptional induction of INO1
. The evidence that speaks to these various points is as follows.
First, we demonstrate that a biochemical signature of sac1
mutants is a dramatic acceleration in the rate of metabolic flux through the CDP-choline pathway for PtdCho biosynthesis (Figure ). This effect is observed for all sac1
alleles, including Δsac1
, and it is observed in sac1-22
strains only when these strains are grown in the presence of inositol. The significance of the latter point is that sac1-22
strains, although exceptional from the standpoint that these are not inositol auxotrophs, are only able to exhibit bypass Sec14p phenotypes when grown in inositol-containing medium (Kearns et al., 1997
). The signature alterations in inositol phospholipid metabolism characteristic of sac1
strains are also recorded in sac1-22
strains but, again, only when these mutants are provided with inositol in the growth medium (Kearns et al., 1997
; see above).
Second, our demonstration that accelerated rates of CDP-choline pathway activity correlate with bypass Sec14p in sac1 mutants is consistent with the DAG production model shown in Figure . Because sac1 strains exhibited wild-type levels of bulk DAG at steady state, an expected consequence of excess DAG production in sac1 strains would be a compensatory increase in the activity of a DAG degrading–consuming pathway. We conclude that the CDP-choline pathway represents a major metabolic sink for excess DAG in sac1 strains. Our finding that CDP-choline pathway hyperactivity in sac1 strains is sensitive to the metabolic conversion of DAG to PtdOH effected by DGK expression also supports this concept (Figure C). That this excess DAG is ultimately produced from PtdOH, a phospholipid that is a direct product of PLD action, is suggested by the demonstration that PLD inactivation (even when exogenous choline is supplied in vast excess) reduces CDP-choline pathway activity in sac1 strains to essentially wild-type levels (Figure A).
The stimulation of the CDP-choline pathway recorded for sac1
strains presents an intriguing paradox. The essence of the paradox is that, although hyperactivated CDP-choline pathway activity correlates with Sac1p-mediated bypass Sec14p, genetic inactivation of the CDP-choline pathway constitutes a recognized mechanism for bypass Sec14p (Cleves et al., 1991b
). The DAG model illustrated in Figure reconciles this contradiction in a simple manner. It posits that, although increased DAG production effects bypass Sec14p, the resultant consumption of DAG manifests itself in elevated CDP-choline pathway activity. The stimulation of the CDP-choline pathway in sac1
mutants raises the possibility that DAG availability helps set the baseline rate of metabolic flux through this pathway in yeast. Such a DAG effect could potentially be mediated by DAG stimulating the activity of CCTase, the rate-determining enzyme of the CDP-choline pathway.
Third, our results lend insight into the role of PtdIns-4-P accumulation in the sac1
-mediated mechanism for bypass Sec14p. Because PLD-insufficient sac1
strains still accumulate high levels of this phosphoinositide (Figure , A and B), yet are incompetent for bypass Sec14p (Xie et al., 1998
), we conclude that increased PtdIns-4-P is at best a contributing factor to bypass Sec14p. Indeed, the finding that PLD deficiency abolishes the bypass Sec14p phenotype of sac1
strains in the face of PtdIns-4-P accumulation suggests that excess PtdIns-4-P may contribute to bypass Sec14p by effecting a downstream activation of PLD (Figure ). For example, PtdIns-4-P could modulate PLD activity indirectly by influencing the action of another protein whose function is to regulate PLD. The yeast oxysterol-binding protein homologue Kes1p and the BSD1
gene product represent candidate PLD regulators (Figure ), because the bypass Sec14p growth phenotypes of kes1
mutants are also completely abolished by PLD insufficiency (Figure C). Kes1p is a particularly attractive candidate because it binds PtdIns-4-P, and Kes1p overproduction phenocopies PLD inactivation (our unpublished data). Interestingly, CDP-choline pathway mutations exhibit a significant PLD-independent component of their ability to suppress sec14
defects (Figure C). We attribute this PLD-independent component to reflect reduced rates of DAG consumption (Figure ).
Fourth, we report insights into the mechanism that underlies the inositol auxotrophy of sac1 strains. We demonstrate that CDP-choline pathway activity contributes to this inositol requirement. Intereference with the activity of this PtdCho biosynthetic pathway at any one of several points restores the ability of Δsac1 strains to grow in the absence of exogenous inositol (Figure B) at wild-type rates (Figure ). Because sac1-associated inositol auxotrophy does not result in obvious defects in transcriptional regulation of INO1 (Figure , A and B), nor is it accompanied by the classical “inositol-less death” of ino1 mutants (Figure ), we conclude that Sac1p deficiency results in an inability of cells to thrive on endogenously produced inositol. In that regard, the accumulation of PtdIns-4-P in sac1 strains indicates disruption of a substantial metabolic flux from PtdIns-4-P to PtdIns. This block in PtdIns production may contribute to the unusual inositol auxotrophy of sac1 strains in a manner that does not solely operate through CDP-choline pathway hyperactivation.
Finally, we emphasize the pleiotropic nature of the phospholipid metabolic alterations that accompany Sac1p dysfunction. Some of these are involved in mediating bypass Sec14p (i.e., accumulation of PtdIns-4-P and subsequent increases in PLD-mediated DAG production). We propose others to represent indirect correlates of the bypass Sec14p condition (e.g., accelerated CDP-choline pathway activity and reduced PtdSer levels). A continuing challenge in these analyses is the recognition of which alterations in phospholipid metabolism in bypass Sec14p mutants most directly mediate Sec14p-independent growth and Golgi secretory function in yeast.