Sec2p directly binds to PI4P
To explore a possible role of phosphoinositides in Sec2p function, we incubated Sec2p with liposomes containing various phosphoinositides. Liposome-bound Sec2p was precipitated by centrifugation and detected by Coomassie blue staining. Yeast contains four major phosphoinositides marking different compartments: endosomal PI3P, Golgi-associated PI4P, plasma membrane PI(4,5)P2
and vacuolar PI(3,5)P2
(Strahl and Thorner, 2007
). Sec2p showed the highest binding to liposomes containing PI4P (, top panel, lane 1), yet also showed some affinity to PI(3,5)P2
(lane 3 and 4). The binding to PI3P was at the same level as that to control liposomes containing phosphoserine (PS) (compare lane 2 and 5). Sec2 did not precipitate in the absence of liposomes (lane 6). The approximate Kd of Sec2p for PI4P was determined to be 185 μM by incubating equal amounts of GST-Sec2p with various concentrations of liposomes containing PS, PI3P or PI4P (, bottom panel).
Figure 1 (A) Sec2p directly binds to Phosphoinsitides. (top panel) Sec2p-His6 (120 nM) purified from E. Coli was incubated by itself (lane 6) or with 0.5 mM liposomes containing 50/30/15 mol% of PC/PE/PS and 5 mol% of the indicated phosphoinositides (lanes 1–4), (more ...)
Localization of Sec2p requires Pik1 activity
Pik1p is an essential phosphatidylinositol 4-kinase that acts in both the nucleus and the Golgi, and is required for post-Golgi transport (Hama et al., 1999
) (Audhya et al., 2000
; Walch-Solimena and Novick, 1999
). The pik1-101
mutation leads to inactivation of the kinase activity at 37°C, however, slow growth, a reduction of cellular PI4P levels and a partial secretion defect are observed in pik1-101
cells even at 25°C (Walch-Solimena and Novick, 1999
). We analyzed the distribution of Sec2-3xGFP in wild type and pik1-101
yeast cells. The 3xGFP coding sequence was fused to the C-terminus of the SEC2
coding sequence and expressed behind the SEC2
promoter as the sole copy of SEC2
in yeast. Cells were grown at 25°C, shifted to 37°C for 60 min, then fixed with methanol and observed by epi-fluorescence microscopy. In wild type cells, Sec2-3xGFP localized to the tips of small and medium size buds, and to the mother-daughter neck of larger cells at all temperatures (, top four panels). This pattern reflects the association of Sec2p with secretory vesicles concentrated at sites of secretion (Elkind et al., 2000
). Localization of Sec2-3xGFP to sites of secretion was observed in some pik1-101
cells, at 25°C (, third panels from the top), however, the percentage of the cells exhibiting this localization was only half that of wild type cells (, left panel). Following a shift to 37°C, Sec2-3xGFP was largely mislocalized to the cytoplasm (, bottom panels). A small population of Sec2 was mislocalized to puncta that were not positive for a late-Golgi marker Sec7p (not shown). The percentage of cells exhibiting normal localization was very low relative to wild type (, right panel).
To determine if the effect of pik1-101
on Sec2p localization is indicative of a failure in the vesicle association of Sec2p or a failure of vesicles to concentrate at exocytic sites, we examined the localization of Sec6-3xGFP. Sec6p is a component of the Exocyst complex that tethers secretory vesicles to the plasma membrane (TerBush and Novick, 1995
). Most exocyst components, including Sec6p, are associated with secretory vesicles as they move towards the plasma membrane along polarized actin cables (Boyd et al., 2004
). Wild type and pik1-101
cells expressing Sec6-3xGFP as the sole copy were grown at 25°C, shifted to 37°C for 60 min, then fixed with methanol and analyzed by fluorescence microscopy. Sec6-3xGFP localized to the bud tip or mother-daughter neck in wild type cells (, top four panels). Although the expression level of Sec6-3xGFP in pik1-101
was about 64% of wild type, leading to a somewhat weaker fluorescence signal, Sec6-3xGFP localized normally both at 25°C and 37°C (, bottom four panels). The percentage of cells exhibiting normal Sec6-3xGFP localization was reduced to some extent, perhaps reflecting reduced vesicle production, but less severely then Sec2-3xGFP (). Thus, the localization of Sec2p is more sensitive to the loss of Pik1p function than that of Sec6p, suggesting that the association of Sec2p with secretory vesicles is dependent upon the pool of PI4P generated by Pik1p.
Sec2p localization depends on PI4P-binding affinity via its positively charged patches
To determine if PI4P-binding is required for Sec2p localization, we first mapped the PI4P-binding region in Sec2p. Since Sec2p has no conserved lipid-binding motif, various truncation constructs were tested in vitro for binding to PI4P liposomes. Sec2p-truncation mutants consisting of a.a. 1–160, 1–258, 1–374, and 509–759 (C-terminus) showed no specific binding to PI4P, however, a.a. 1–450, 1–508, 1–623, and 450–759 bound to PI4P (not shown), suggesting that the phosphoinositide binding region lies between a.a 374 and 508. Within this region there are three positively charged patches (). Mutation of positively charged amino acids to alanine in patch 1, 2, or 3 individually had only a slight effect on PI4P-binding (). Combinations of mutations in patch 1 and 2, or patch 2 and 3, reduced PI4P-binding affinity to about 30% of the wildtype (). When all three patches were mutated, PI4P-binding was reduced to about 10% of wildtype (), indicating that all three patches are involved in PI4P binding. Hereafter we refer to the mutant that has mutations in all three patches as the Sec2-KA mutant.
Sec2p-PI4P interaction is required for Sec2p localization
To test the localization of Sec2-KA, the mutations were introduced into the SEC2 locus and the 3xGFP sequence was fused to the C-terminus. The localization of Sec2-KA-3xGFP was significantly affected (, left panels). Localization to the bud tip or bud neck was not seen in most cells, however, ~30% of the cells exhibited normal localization, but with reduced intensity (, left bottom, arrow). This result indicates that the PI4P-interaction is important for Sec2p localization.
Sec2p transiently associates with PI4P-enriched membranes
To determine if Sec2p localizes to sites of PI4P concentration, we generated a strain expressing both Sec2-3xGFP and mCherry-tagged to the Pleckstrin homology (PH) domain of the FAPP1 protein. This domain shows a high specificity for PI4P in vitro (Dowler et al., 2000
) and several groups have shown that the FAPP1-PH domain recognizes PI4P in yeast, even though FAPP1 is a mammalian protein (Stefan et al., 2002
) (Roy and Levine, 2004
) (Faulhammer et al., 2005
). The mCherry sequence was attached to the N-terminus of the FAPP1-PH domain and this fusion protein was expressed behind the ADH1
promoter. The mCherry-FAPP1-PH signal was lost when cells were fixed in methanol, so we observed live cells with a spinning disk confocal microscope. FAPP1-PH was distributed throughout the cells, yet showed accumulation on punctate structures (, middle) that have been shown to be elements of the Golgi apparatus (Levine and Munro, 2002, Stefan et al., 2002
, Faulhammer et al., 2005
, (Tahirovic et al., 2005
). The most prominent concentration of Sec2-3xGFP localized to the bud tip and mother-daughter neck and did not colocalize with FAPP1-PH (, left panel). Nevertheless, a portion of Sec2-3xGFP localized to small puncta that might represent secretory vesicles or elements of the Golgi. About 43% of the Sec2-3xGFP puncta were also positive for FAPP1-PH and similarly about 38% of the FAPP1-PH puncta were also positive for Sec2-3xGFP (, arrowheads, inset). This result suggests that Sec2p transiently associates with sites of high PI4P concentration on the Golgi, yet PI4P levels are reduced once secretory vesicles arrive at sites of secretion.
The growth defect of pik1-101 is partially suppressed by Sec2 overexpression
The growth defect of pik1-101
might be due in part to Sec2p mislocalization, which would in turn lead to a failure in Sec4p activation. In this case, restoring Sec4p activity by either overexpressing its activator, Sec2p, or by directly overexpressing Sec4p, might be expected to at least partially rescue the growth defect of pik1-101
cells. In addition, our prior studies demonstrated that overexpression of Ypt32p could restore the localization of two Sec2p mutants (Ortiz et al., 2002
). Therefore, Sec2p localization might be restored in pik1-101
by overexpressing Ypt32p, which would rescue the growth defect of pik1-101
cells. To test these possibilities, Sec2p, Sec4p, and Ypt32p, were overexpressed from high copy number plasmids in a pik1-101
strain. As shown in , wild type cells grew at all temperatures, whereas pik1-101
could not grow well above 30°C. When Sec2p, Sec4p, or Ypt32p were overexpressed, pik1-101
cells were able to grow well at 30°C (, middle panel), and grow slowly at 35°C (, right panel). At 37°C, no growth was evident, indicating that the rescue of pik1-101
was only partial (not shown). In contrast, overexpression of Ypt1p, the rab GTPase that acts early in the secretory pathway, did not rescue the pik1-101
growth defect at any temperature tested. The partial nature of the rescue of pik1-101
by overexpression of Ypt32p, Sec2p or Sec4p indicates that PI4P must have another essential function(s) in addition to the recruitment of Sec2p. In fact, Pik1p has recently been implicated in the recruitment of a lipid flippase and a PI4P-binding protein required for budding of vesicles from the Golgi (Dippold et al., 2009
; Natarajan et al., 2009
Overexpression of Sec2p, Ypt32p and Sec4p partially suppresses the growth defect of pik1-101
To determine if Ypt32p overexpression restores Sec2p localization in pik1-101
cells, we first confirmed that Ypt32 overexpressed from a high copy number plasmid rescued the growth defect of pik1-101
cells expressing Sec2-3xGFP at 30°C (Supplemental Figure S2
). Next, we observed the localization of Sec2-3xGFP in cells overexpressing Ypt32p. In wildtype cells, overexpression of Ypt32p did not affect the localization of Sec2-3xGFP at either permissive (not shown) or restrictive temperature (, top panels). In pik1-101
cells Sec2-3xGFP was mislocalized at the restrictive temperature (, third panel from the top), however, when Ypt32p was overexpressed, Sec2p-3xGFP localization was largely restored (, bottom panel; ), particularly in budded cells.
Both PI4P-binding and Ypt31/32p are required for growth
The restoration of Sec2p localization in pik1-101
cells by Ypt32p overexpression, suggests that Ypt32p and PI4P might act in parallel to control Sec2p localization. PI4P-binding deficient sec2
mutant cells (sec2-KA
) did not show a growth defect at any temperature tested (not shown), thus we determined if there were synthetic effects when Ypt32 and PI4P-binding deficient mutants were combined. Since Ypt31 and Ypt32 are essential for growth, yet functionally redundant (Benli et al., 1996
), we used a combination of mutations, ypt31Δ ypt32A141D
, to disrupt their function. The ypt31Δypt32A141D
strain exhibits temperature sensitive blocks in growth and secretion (Jedd et al., 1997
). Diploids carrying the sec2-KA
alleles were sporulated and the resulting tetrads were dissected at 25°C. Spores carrying the ypt31Δ ypt32A141D
alleles grew normally at 25°C (, circle), 30°C, and 32°C (not shown). In contrast, spores expressing sec2-KA
and ypt31Δ ypt32A141D
alleles grew extremely slowly at 25°C (, square), 30°C, and 32°C (not shown). Neither double (ypt31Δypt32A141D
) nor triple mutants grew at 37°C (not shown). Localization of the Sec2-KA protein in the ypt31Δypt32A141D
background was almost completely abolished (not shown). These results are consistent with a model in which PI4P and Ypt31/32p act in parallel to regulate Sec2p function.
Sec2p localization is unaffected in a PI4P phosphatase mutant
Sac1p is a phosphatidylinositol phosphate phosphatase that acts on phosphoinositides including PI4P (Guo et al., 1999a
) (Hughes et al., 2000
). Inactivation of Sac1p leads to a large increase in the PI4P concentration of the ER and the Golgi membrane (Foti et al., 2001
). To test whether the localization of Sec2p is affected by a shift in the distribution of PI4P, we first examined PI4P localization using the GFP-tagged FAPP1-PH domain construct in live sac1-6
cells at 30°C. While GFP-FAPP1-PH was highly concentrated on puncta in wild type cells, in sac1-6
cells GFP-FAPP1-PH localized to ring shaped structures typical of the nuclear envelope and cortical ER (Supplemental Figure S1A
). Puncta were observed in sac1-6
cells, however they were not enhanced, suggesting that PI4P levels were increased most prominently in the ER rather than the Golgi. We next examined the localization of Sec2-3xGFP and found that it localized normally in sac1-6
cells (Supplemental Figure S1B
). Thus, although Sec2p binds PI4P in vitro and Sec2-3xGFP requires Pik1p function for localization in vivo, Sec2-3xGFP does not shift localization solely in response to a shift in the distribution of PI4P.
PI4P and PI(4,5)P2 at the plasma membrane are not required for Sec2p localization
In yeast, another essential PI4-kinase, Stt4p, generates an independent pool of PI4P at the plasma membrane (Audhya and Emr, 2002
). Mss4p, a PI4P5-kinase, then utilizes the PI4P produced by Stt4p, to generate PI(4,5)P2
at the plasma membrane (Homma et al., 1998
) (Desrivieres et al., 1998
). Sec2-3xGFP localized normally in stt4-4
cells at both the permissive temperature 25°C (not shown), and following a shift to 37°C for 60 min (Supplemental Figure S1C
, top panels). Sec2-3xGFP also localized normally in the mss4-102
mutant at both 25°C (not shown) and following a shift to 37°C for 60 min (Supplemental Figure S1C
, bottom panels). These results indicate that Sec2p localization is independent of the PI4P and PI(4,5)P2
pools at the plasma membrane.
PI(3,5)P2 on the vacuolar membrane is not required for Sec2p localization
In vitro liposome-binding experiment showed that Sec2p has significant affinity to PI(3,5)P2
(). To test if Sec2p localization is regulated by PI(3,5)P2
, we examined the localization of Sec2p-3xGFP in a fab1Δ
, PI3-5 kinase deletion mutant and found that it was unaffected (Supplemental Figure S1D
, bottom panels).
Two-hybrid screen for Sec2p mutants that no longer bind Ypt32p
Overexpression of Ypt32p restores the localization of Sec2p in pik1-101
cells (), and in two Sec2p mutants, sec2-59
(Ortiz et al., 2002
), suggesting that Ypt32p acts together with PI4P in Sec2p membrane recruitment. To critically evaluate this proposal, we sought sec2
mutations that lost their interaction with Ypt32p, but not with Sec15p or PI4P. We used the yeast two-hybrid system to screen for Sec2p mutants that had lost the ability to interact with Ypt32p. SEC2
were cloned into the GAL4 activation domain vector pACTII and GAL4 DNA-binding domain vector pGBKT7, respectively. PCR random mutagenesis was performed on the 5′ end of SEC2
(codons 1–311). The mutagenized sec2
sequences were cloned back into pACTII and co-transformed with YPT32
into yeast. Co-transformants were screened for loss of interaction as determined by their inability to grow on either SC-his plates, or SC-his with 5 mM 3-amino-1,2,4-triazole (3-AT), a competitive inhibitor of His3p function. A total of six sec2
mutants were identified that showed at least some reduction in His3p function relative to wild-type. Three were shown to be -His and three were +His, yet unable to grow in the presence of 3-AT (Supplemental Figure S3A
). All clones identified as having lost interaction with Ypt32p were further screened to confirm that Sec2p was still full length by immunoblot analysis using an anti-HA antibody that recognizes the N-terminal HA epitope on Sec2p (not shown).
The Ypt32p-binding domain of each sec2
mutant was sequenced and aligned with the wild-type sequence (Supplemental Figure S3B
). The level of mutagenesis ranged from one amino acid change (M13 and M14) to five (M7). The sec2
mutant M19 has two changes and both M5 and M37 have three. Although the level of mutagenesis and screening did not saturate the Ypt32p-binding domain, the amino acid changes are clustered in two general areas.
Sec2p mutants have decreased affinity for Ypt32p-GTP in vitro
The region encoding the Ypt32p-binding domain of each sec2 mutant (codons 157–339) was sub-cloned into a GST-Sec2p expression construct. The GST-Sec2 mutant proteins were purified from E. coli on glutathione beads and used in binding assays with purified, recombinant His6-Ypt32p pre-loaded with GTPγS. The amount of Ypt32p bound was evaluated by immunoblot using an anti-Ypt32p antibody. Wild-type Sec2p bound approximately 10% of the available Ypt32-GTP under these conditions (). Greatly reduced Ypt32-GTP binding was seen with four of the six mutants identified in the two-hybrid screen (M7, M5, M14, and M37). A longer exposure of the Western blot shows that these mutants still have some affinity for Ypt32p. The M13 sec2 mutant, although identified in the two-hybrid screen as having a weakened interaction with Ypt32p, bound levels similar to wild type in vitro. M19, which was identified as having no interaction in the two-hybrid system, bound approximately 50% of wild type levels in vitro. Since only a portion (codons 157–339) of the mutagenized region of SEC2 (codons 1–311) was transferred into the expression construct, it is possible that a mutation upstream of the sub-cloned region contributed to the loss of interaction observed in the two-hybrid screen with M13 and M19.
Sec2p-Ypt32p interaction is required for Sec2p localization, growth, and secretion
Sec2p mutants have increased affinity for Sec15p, and normal affinity for PI4P
The Sec2p mutants were next tested to see if their affinity for Sec15p had also been altered. We used both His6
-tagged full-length Sec15 protein (Sec15-FL) and a C-terminal fragment (Sec15-C, a.a. 559–910) since the full-length Sec15p is more difficult to express and purify in large amounts (Medkova et al., 2006
), and it interacts less efficiently than the C-terminal portion of the protein. We observed a very striking inverse correlation between Ypt32p-binding and Sec15p-binding. Those mutants that exhibited decreased binding to Ypt32p (M7, M5, M14 and M37), exhibited increased binding with both full-length Sec15p and the C-terminus of Sec15p ().
We next tested the effects of the mutations on the interaction of Sec2p with liposomes containing PI4P. All mutant proteins showed near normal association with liposomes containing PI4P and this level of binding was significantly greater than that seen with control liposomes containing PS instead of PI4P (Supplemental Figure S3C
). Thus, these mutations do not interfere with PI4P binding.
The Sec2p-Ypt32p interaction is needed for localization of Sec2p, but not Ypt32p
The four sec2
mutants that showed greatly reduced binding to Ypt32p were tagged with 3xGFP and introduced into yeast as the sole copy of SEC2.
An immunoblot using anti-Sec2p antibody shows that the levels of Sec2-3xGFP and the four, tagged mutant proteins are similar to that of endogenous Sec2p (Supplemental Figure S3D
). Yeast strains were grown overnight at 25°C, harvested, washed, and visualized by fluorescence microscopy. Sec2-3xGFP was localized in small buds and mother-daughter necks of wild-type cells. All four Ypt32-binding-deficient Sec2p mutants showed diffuse fluorescence throughout the cell with little or no apparent concentration at bud tips and necks (; quantification shown in Supplemental Figure S3E
While these results confirm that the localization of Sec2p requires its interaction with Ypt32p, we next asked if Ypt32p localization requires its interaction with Sec2p. An mCherry-Ypt32p construct was introduced at the URA3 locus of each of the Sec2-3xGFP strains. Ypt32p was localized normally in all strains, even though it has greatly reduced affinity for the mutant Sec2 proteins (, arrowheads). Thus, the localization of Ypt32p is not dependent on its interaction with Sec2p.
The Sec2p-Ypt32p interaction is needed for growth and secretion
To determine if the loss of the Sec2p-Ypt32p interaction has an effect on growth, serial dilutions of yeast strains containing Sec2-3xGFP, Sec2-78, or each of the M-mutants tagged with 3xGFP were spotted onto YPD plates and incubated at either 34 or 37°C (Supplemental Figure S3F
). M7, M5, M14 and M37, although not completely dead at 37°C, as was the negative control sec2-78
, were severely impaired. Overexpression of Ypt32p completely rescued the growth defects of these sec2
mutants and restored the localization of the mutant proteins (Supplemental Table SI
We then assayed the mutant strains for their ability to secrete the cell wall enzyme invertase. As shown in , wild type secretes nearly all of the invertase made during a one hour period of derepressed synthesis. The mutants deficient in binding Ypt32p showed a block in secretion comparable to that of sec2-78.
Ypt32p-binding deficient sec2 mutants show a different conformation
The Sec2-78p mutant binds Sec15p more tightly than wild type and exhibits a different limited proteolysis pattern when incubated with trypsin, suggesting that it exists in an alternate conformation (Medkova et al., 2006
). Since the Sec2 mutants that have lower affinity to Ypt32p show phenotypes similar to Sec2-78p (), we examined whether those mutants proteins have a conformation similar to Sec2-78p. GST-tagged wildtype Sec2 and each of the mutants were purified from yeast, and incubated with trypsin. The limited proteolysis pattern was detected with anti-Sec2 antibody that recognizes the N-terminal region. Wildtype Sec2p accumulated a major degradation product of ~70 kDa (). In contrast, the major degradation product of the Sec2 mutants was ~50 kDa and the overall proteolysis pattern was distinct from that of wildtype (). Since the pattern was similar to that of Sec2-78p, the new Sec2 mutants might have conformations similar to that of Sec2-78p despite the different sites of these mutations.
Ypt31p and Ypt32p are required for Sec2p localization
We next tested Sec2p localization in cells deficient for Ypt31p and Ypt32p. Cells were grown overnight at 25°C, fixed, and visualized by fluorescence microscopy. In ypt31Δypt32A141D cells, Sec2-3xGFP showed diffuse fluorescence throughout the cell with no concentration at the sites of secretion, even at the permissive temperature (). This result confirms that Ypt31 and Ypt32 are required for Sec2p localization.
PI4P does not inhibit the binding of Sec2p to Ypt32p
To explore the possibility that PI4P binding influences the ability of Sec2p to interact with Ypt32p, we have performed in vitro binding studies. His6
-Sec2p was pre-incubated in the presence or absence of liposomes containing either PS or PI4P. GST-Ypt32p was immobilized on beads and preloaded with GDP or GTPγS, and the Sec2p-liposome complex was added to the beads. After the reaction, the binding buffer and unbound proteins were removed from beads, which were then washed with buffer containing Triton X-100. Detergent was included in the wash buffer to prevent liposome-mediated precipitation. Sec2p bound more efficiently to GTPγS-loaded Ypt32p, as previously shown (Ortiz et al., 2002
). Neither control liposomes nor PI4P liposomes had a significant effect on the binding of Ypt32p to Sec2p ().
Three-way interaction between Sec2p, PI4P and Ypt32-GTP
Sec2p, Ypt32p and PI4P can form a ternary complex
The localization studies suggest that Ypt32p and PI4P act in parallel in Sec2p recruitment (, and ). These observations prompted us to ask whether Sec2p can bind Ypt32p and PI4P at the same time. To address this question, we performed in vitro liposome binding experiments. GST-Sec2p, GST-Ypt32p, or GST were purified from E. Coli and eluted from Glutathione beads. Ypt32p was preloaded with either GDP or GTPγS. GST-Sec2p, nucleotide-loaded GST-Ypt32p, or GST were incubated with liposomes containing PI4P. Liposome-bound proteins were precipitated by centrifugation and analyzed with anti-GST antibody. Only trace amounts of Ypt32p were precipitated with PI4P-liposomes in the absence of Sec2p (, lane 3,5), indicating that Ypt32p does not have an ability to directly bind PI4P. However, in the presence of Sec2p, Ypt32p was precipitated with PI4P-liposomes, more efficiently in its GTP-bound form (, compare lane 4 and 6). This result implies that Sec2p, Ypt32p and PI4P can form a ternary complex.
PI4P inhibits the binding of Sec15p binding to Sec2p
We next tested if PI4P-binding influences the ability of Sec2p to interact with Sec15p in vitro. GST-tagged Sec2p was immobilized on beads, pre-incubated in the presence or absence of liposomes containing either 50 μM of PS, or 10 to 50 μM of PI4P. Sec24p, which is similar in size to Sec2p, was used as a negative control. Bacterially purified His6-Sec15p was added to the Sec2p or Sec24p-liposome mixture, and bound complex was pulled down with Glutathione beads. After the reaction, the beads were washed with buffer containing Triton X-100. Detergent was included in the wash buffer to prevent liposome-mediated precipitation of Sec15p. Since Sec15p binds both control liposomes and PI4P liposomes (not shown), if the experiment were done in the absence of detergent, GST-Sec2p would pull down PI4P liposomes which would then pull down Sec15p regardless of its ability to directly bind Sec2p. Sec2p bound Sec15p and control liposomes containing PS had little effect on the binding (, lane 4 and 5). PI4P liposomes strongly inhibited the Sec15p-Sec2p interaction in a dose dependent manner (, lane 6–9). GST-Sec24p did not bind to Sec15p (, lane 1–3).
PI4P inhibits binding of Sec15p to Sec2p
To determine if PI4P affects the binding affinity between Sec2p and Sec15p, various concentration of GST-Sec2p immobilized on beads were pre-incubated with liposomes containing PS or PI4P, and then purified His6 -Sec15p was added to the beads. As shown in , control liposome containing PS did not affect the Sec2-Sec15 interaction, however, PI4P significantly reduced the affinity between Sec2p and Sec15p over a range of Sec2p concentrations ().
The Sec2p-Sec15p interaction is enhanced in a pik1-101 mutant
To test the effect of PI4P on the interaction of Sec2p with Sec15p in a more physiological setting we compared the efficiency of co-precipitation of Sec15-13xMyc with Sec2-GFP from pik1-101 mutant and wild type lysates. Cells were grown overnight at the permissive temperature and Sec2-GFP was precipitated from lysates with anti-GFP antibody. As shown in , significantly more Sec15-13xMyc was co-precipitated with Sec2p from a pik1-101 lysate than from a wild type lysate, although this increase was not as dramatic as the increase seen in a sec2-78 lysate. Cytosolic and membrane-bound pools of Sec2p were not resolved in this experiment. This result supports the hypothesis that the PI4P made by Pik1p acts to inhibit the binding of Sec15p to Sec2p.