Formation and extrusion of TVS mediated by overexpression of MLN1
During prolonged observation of oocytes expressing MLN1, we witnessed phenomena that suggested a potential link between the activity of MLN1 and the processes of membrane reshaping and vesiculation. To avoid potential deterioration in the integrity of oocytes, most of our previous electrophysiological studies [1
] were carried out 2-3 days after injection of the oocytes with MLN1-cRNA. Many oocytes, however, remain viable for extended periods, and when these were examined we noticed an increasing number of tubular and vesicular structures extruding from apparently intact oocytes (). These TVS began appearing 3-5 days after injection with MLN1-cRNA, and later there was a dramatic increase in their numbers. Small vesicular buds or bulb-shaped formations can be seen on the surface of oocytes on days 3-5 (), and these protrusions grow into cylindrical and twisted tubular structures by days 6-8 (). After 6 days, TVS of various sizes surround the oocytes. Some of them are smaller than 1 μm, while others are 10-30 μm or even larger in diameter (). They appear to be pinched off from the tubules that are formed initially or to have been released from the oocyte in a manner analogous to the exocytosis of multi-vesicular bodies. They are similar in shape but larger in size than the exosomal, megavesicular, tubular and other types of TVS [28
] that are related to LE/L-dependent secretory pathways. TVS were also abundantly formed in MLN1-expressing oocytes treated with actinomycin D, an approach commonly used to distinguish between the activities mediated by exogenously expressed proteins and those arising from endogenous channels or other proteins in Xenopus
oocytes (data not shown). No such structures were observed in control, H2
O-injected oocytes ().
Tubulo-vesicular structures extruded from oocytes overexpressing wild type (WT) MLN1
The serine lipase active site of MLN1 is required for its ability to generate tubulo-vesicular structures
Given the known cation channel activity of MLN1, we initially surmised that over-expression of this protein was causing a disturbance in the transmembrane gradients for Ca2+
and/or other cations, which caused osmotic stress and resulted in the formation of membrane blebs and vesicles. However, we did not observe the formation of similar structures in oocytes expressing other Ca2+
-permeable cation channels of the TRP family, e.g., TRPP2, TRPPL, TRPV5 and TRPV6, all of which have large Ca2+
Since MLN1 contains a consensus sequence for a serine lipase active site in the large loop between the first and second transmembrane domains (), we considered the possibility that this site is functional and may play a role in the action of MLN1 on membrane reshaping. For this purpose, we constructed an MLN1 mutant containing a modified, presumably inactive (see below) serine lipase active site (SL-MLN1). Visual comparison of oocytes injected with the cRNAs for the mutant SL-MLN1 or WT-MLN1 under the same conditions showed that no TVS were formed in the oocytes expressing SL-MLN1 (). However, they were abundantly generated in the oocytes expressing WT-MLN1 (Figures ; and ). Fluorescent subcellular clusters containing the GFP-tagged SL-MLN1 are evident in the subcortical regions of oocytes expressing this mutant (). These clusters, however, do not lead to budding and extrusion of TVS as seen in the WT-MLN1-expressing oocytes. These data suggest that the serine lipase site of MLN1 is essential for formation of the TVS (). Examination of a twisted tubular formation that was extruded from an oocyte expressing GFP-tagged WT-MLN1 shows that MLN1 is predominantly located in the leading edge of the tubule (). Furthermore, there are several striations of GFP fluorescence in this area, with indentations on the sides of the tubular structure, perhaps illustrating the initial points at which vesicles are pinched off from the tubule. shows a larger area of a different oocyte where the GFP-tagged MLN1 is again mainly localized near the advancing edge of a large protrusion toward the upper right side of the field, and a non-specific red autofluorescence becomes apparent in the lower area on the opposite end of the oocyte. No such TVS are observed in oocytes expressing GFP-tagged SL-MLN1 () or in H2O-injected oocytes where only the red autofluorescence is evident (). shows that the percentage of oocytes producing TVS markedly increases after day 2 in both actinomycin D-treated (ML+ACT) and untreated oocytes expressing WT-MLN1 (ML) (p < 0.05 compared with the corresponding basal value), while no such structures were observed in H2O-injected oocytes (H2O) or in oocytes expressing the SL-MLN1 mutant (SL). We found similar levels of GFP fluorescence intensity after injection of oocytes with WT-MLN1-GFP or SL-MLN1-GFP cRNAs (), indicating that the membrane reshaping effects that we observed were not merely due to higher expression of the WT-MLN1 versus the SL-MLN1 proteins in the respective batches of oocytes.
Formation of TVS is deficient in oocytes expressing MLN1 with a mutated serine lipase active site (SL-MLN1)
Mutation of the serine lipase active site abolishes the phospholipase activity
Since MLN1 is a membrane protein integrated into the phospholipid bilayer, we explored the possibility that it may act as a phospholipase (PL). Based on the evidence that the serine lipase site in MLN1 plays a role in membrane remodeling, we set out to define the type of lipase activity mediated by this protein. We utilized fluorescent probes for phospholipase A1
) and obtained evidence indicating that wild type MLN1 can mediate phospholipase A (PLA). After homogenizing the oocytes, we tested their PLA activities using the bis-BODIPY FL C11
-PC probe [20
] for PLA1
, which was used to study the functional expression in Xenopus laevis
oocytes of another recently cloned lysosomal PLA2 [21
]. In this latter study on whole oocyte homogenates, an increase in PLA2 activity of more than 40% above the background level (H2
O-injected oocytes) was measured with this probe in oocytes expressing PLA2, similar to the values we observed on day four in oocytes expressing WT-MLN1 (). We also used a probe that is specific for PLA2, Bodipy FL C5
–HPC, and obtained similar results. The PLA activity in WT-MLN1-expressing oocytes increased considerably several days after injection of the oocytes with WT-MLN1 cRNA (p < 0.05 compared with the corresponding basal value), while no substantial change in the background enzymatic activity occurred in the H2
O-injected oocytes (). The PLA activity in SL-MLN1-expressing oocytes was not increased significantly above that in the H2
O-injected oocytes. Thus the increase in PLA activity of WT-MLN1 occurring several days after injection () corresponds to, and even precedes, the accumulation of TVS (). These results, together with the lack of significant changes in the H2
O-injected oocytes and in those expressing mutant SL-MLN1, suggest that the PLA activity of MLN1 plays a key role in the formation of these structures.
Comparison between the enzyme and channel activities and the contribution to lysosomal exocytosis of WT-MLN1 and SL-MLN1 mutant
Similar channel activities of WT-MLN1 and SL-MLN1
We considered the possibility that the formation of the TVS is mediated not directly by the PLA activity but by changes in the MLN1 channel activity when its PLA activity is modulated. Therefore, we studied the channel properties of mutant SL-MLN1 in vesicles prepared from lysosomal membranes after their reconstitution by incorporation into liposomal membrane systems. We did not observe substantial differences between the channel characteristics of the SL-mutant () and WT-MLN1. The Ca2+ and other cation conductances of SL-MLN1 were not significantly different from those of the WT-MLN1 (). The probability of channel opening (nPo) of both SL-MLN1 and WT-MLN1 gradually increased in vesicles prepared on subsequent days after injection of the oocytes (), apparently due to enhanced levels of expression and higher channel densities in the membranes. The increase in nPo of SL-MLN1 was not significantly different from that of WT-MLN1 (). This increase in nPo does not correspond to the insignificant change in PLA activity of SL-MLN1 () and, therefore, cannot explain the lack of formation of TVS in and around the SL-MLN1-expressing oocytes. Despite the lack of substantial differences between the conductances and other channel characteristics of WT- and SL-MLN1 we cannot exclude indirect effects via some binding partners dependent on Ca2+ and other cations on the MLIV-related phenotypes.
The MLN1-stimulated exocytotic trafficking of lysosomes depends on its serine lipase active site
The sequence of events leading to the morphological changes described above is likely to include the translocation of lysosomes toward the cell periphery, resulting in fusion with the cell membrane and release of the lysosomal contents during exocytosis. We have previously shown lysosomal exocytosis to be impaired in human fibroblasts derived from MLIV patients [18
], but to be rescued after transfection with WT-MLN1. To assess lysosomal exocytosis in oocytes, we measured the amount of N-acetyl-beta-D-glucosaminidase (NAG) released by treatment with ionomycin, which promotes the Ca2+
dependent fusion of lysosomes with the PM (). NAG is a water-soluble, lysosome-specific enzyme in the lysosomal lumen whose activity has previously been characterized in Xenopus
]. The NAG assay has principally been used in mammalian cells as a useful test for lysosomal exocytosis, and it has been shown that it is a ubiquitous phenomenon that occurs not only in specialized secretory cells but also in many other types of cells [16
]. We found that the release of this enzyme from WT-MLN1-expressing whole oocytes is significantly higher than that from H2
O-injected (control) and SL-MLN1-expressing oocytes () (p<0.05 for WT vs. control and WT vs. SL), particularly following stimulation with the Ca2+
ionophore, ionomycin. There was no statistically significant difference between the NAG release from the SL-MLN1-expressing oocytes and that from the H2
O-injected oocytes. These findings suggest that WT-MLN1 promotes the exocytotic trafficking of lysosomes, especially at higher levels of cytosolic Ca2+
after the application of the Ca2+
ionophore, and that its role in this process depends to a great extent on its serine lipase site.
Inhibitors of PLA2 impede the MLN1-mediated formation of TVS
To further characterize the PLA activity of MLN1, we studied the effects of various types of phospholipase inhibitors. An inhibitor of the secreted form of PLA2, bromo-phenacyl bromide (BPB), and another PLA2 inhibitor, aristolochic acid (AA), significantly reduced the enzyme activity associated with the expression of MLN1, while inhibitors of phospholipase C (PLC) and phospholipase D (PLD) did not produce substantial reductions ().
To assess the contribution of the PLA activity to the generation of TVS, we tested the actions of the phospholipase inhibitors on this phenomenon. The formation of these structures was inhibited in WT-MLN1-expressing oocytes treated with the PLA2 inhibitors, BPB and AA. We assessed the gradual increase in the percentage of oocytes with associated TVS and found that BPB and AA inhibited the generation of these structures in WT-MLN1-expressing oocytes on day 8 by 48% and 56%, respectively, while the PLC and PLD inhibitors had no significant effect (). The time-course of the formation of these structures is similar to that of the increase in PLA activity of WT-MLN1 (), and their reduction by PLA2 inhibitors corresponds to the antagonistic effects of these inhibitors on the MLN1-associated PLA activity ().
Generation of TVS in vitro using a model membrane system is dependent on the serine lipase site of MLN1
To further explore the role of MLN1-associated PLA activity in membrane remodeling, we carried out studies using in vitro translated MLN1 fragments and a model membrane system. We expressed the soluble fragment spanning the lumenal loop of MLN1 that contains the serine lipase active site (See ). The fragment was expressed as a fusion protein that contained a His-tag for purification and an X-press epitope for identification by Western blot (). We also expressed a corresponding SL-MLN1 fragment. We used thin layer chromatography (TLC) to characterize the PLA2 activity associated with MLN1. We measured the amounts of the fluorescent products formed during incubation with WT-MLN1 and SL-MLN1 protein extracts and confirmed that the WT-MLN1 extract with its intact serine lipase active site generated the expected products of PLA2 activity, while the SL-MLN1 fragment failed to elicit such an effect (). The PLA2-associated activity of WT-MLN1 was reduced significantly by the PLA2 inhibitors, BPB and AA (). The effects of the WT lipase fragment on various substrates were compared. No significant differences were found for the following substrates: bis-Bodipy FL C11
-PC (0.814+/−0.07,+/−SEM, n=5), Bodipy FL C5
-HPC (0.906+/−0.082,+/−SEM, n=5), and PED6 (0.871+/−0.065,+/−SEM, n=5). Both bis-Bodipy FL C11
-PC and Bodipy FL C5
-HPC are phosphatidylcholines, but the chain length of the cleavable fluorescent fatty acid of Bodipy FL C5
-HPC is shorter than that of bis-Bodipy FL C11
-PC. PED6 is a different type of phospholipid, namely a phosphatidylethanolamine [20
]. These results suggest that the PLA2 activity associated with MLN1 is similar for fatty acids of varying chain lengths, and for different types of phospholipids. The pH dependence of the enzymatic activity of MLN1 was weak. At pH 7.4, the activity was 0.688+/−0.075 (+/−SEM, n=5), only 21.8% lower than that at pH 5 (0.906+/−0.082, +/−SEM, n=5).
Phospholipase activity of MLN1 and evidence showing that the serine lipase site of MLN1 is required for the MLN1-mediated tubule formation from model membrane vesicles
We were also able to reproduce the effects of WT-MLN1 on membrane reshaping generated in vitro using a model membrane system. We found that the in vitro translated lumenal loop fragment of WT-MLN1 mediated the formation of tubule-vesicular structures from liposome vesicles (), while the loop fragment of the SL mutant did not exert such an action (). In studies on liposomes containing different fluorescent probes and phospholipids, we found that the WT-MLN1 fragment mediated the formation of long and short tubules resembling cell extensions and multiple fusing vesicular structures (), while the SL-MLN1 fragment failed to generate such structures (). Both of the PLA2 inhibitors, BPB and AA, reduced significantly the formation of tubules as shown by our quantitative analysis (). The values for tubule formation after 2h and 4h incubation in the presence of the WT-MLN1 fragment were 21.8+/−3.12% (n=4), and 73.6+/−5.17% (n=4), respectively. They were normalized in relation to the value observed after 5h incubation (100%). The formation of tubules at pH 7.4 (81.24+/−5.69%, n=5) was only slightly lower (18.76%) than that at pH 5 (100%, value for normalization). The results obtained using the model membrane system suggest that the serine lipase active site on the loop fragment of MLN1 is associated with PLA2 activity and further, that this serine lipase domain is directly involved in membrane vesicular remodeling and potentially, in the growth of tubular structures.
MLN1 mediates TVS formation on the surface of human fibroblasts
The role of the serine lipase domain in mammalian cells should be explored. A patient has recently been identified with a point mutation in the serine lipase active site of the MCOLN1 gene, resulting in an amino acid substitution, 106/Leu→Pro (L106P) in the MLN1 protein [19
]. Although we stop short of showing that the serine lipase domain on MLN1 directly mediates PLA2 activity, collectively these results demonstrate that the PLA2 activity associated with MLN1 plays a role in membrane reshaping. Our experiments were performed using semi-purified fusion proteins from E. coli
and in vitro translated products formed in rabbit reticulocyte lysates. Both of these systems could potentially be contaminated with exogenous PLA2 enzymes. However we would expect that any such contaminating PLA2 activity would be equally represented in both the WT and SL preparations. In light of the possibility that the membrane reshaping that we observed in these artificial systems was the indirect result of the contamination by a nonspecific PLA2, we sought to determine whether MLN1 could be associated with similar phenomenon in vivo
, in a mammalian cell.
We examined the cell surface topology of human primary dermal fibroblasts. We stained the surface of the fibroblasts with CMDiI and a variety of other fluorescent agents and viewed them at 400x magnification. We observed a number of enlarged bulb-shaped TVS protruding from the upper surface and from the ends of the processes of nearly every cell in the normal fibroblast population (). When counterstained with lysosomal dyes we found that these TVS frequently contained LE/L organelles. shows a cluster of intracellular orgenelles stained with LysoTracker® Red DND-99, inside a process that is also labeled with the green cytosolic dye, calcein. We used the same cytosolic probe in combination with another red fluorescent probe, Red DiI-C16(3), which preferentially stains membranes from the LE/L pathway [33
] and observed a similar pronounced distribution of intracellular LE/L organelles along a cellular process and often near the plasma membrane (). The localization of LE/L organelles inside the extensions suggests the involvement of LE/L membrane components in their functions and reshaping. We explored the distribution of GFP-tagged MLN1 expressed in normal fibroblasts. Although the GFP-tagged WT-MLN1 is predominantly localized within the perinuclear region in the interior of the cell (not shown), we found that a substantial amount of MLN1 is located near the cell surfaces as well as in cellular processes (). We often observed bulb-shaped TVS (examples marked with arrow heads in ) in normal fibroblasts, along the processes or at their tips (). shows that the MLN1 is also located in densely packed patches on the plasma membrane of the bulb-shaped TVS. These patches may represent the sites where the LE/L organelles have merged with the plasma membrane.
TVS on the surface of human fibroblasts
The lipophilic fluorescent probe, DiIC16
(3) can be used as a lipophilic marker of the lumenal leaflet of the LE/L membrane because it is not prone to flip-flop from the lumenal to the cytosolic leaflet of the bilayer [33
]. We found that after internalization and intracellular trafficking, a small percentage of the probe is translocated out to the plasma membrane of normal fibroblasts within a few hours, with a concentration in the surface membrane of the filapodia and filapodia-like processes and bulb-shaped TVS (). Fluorescent staining can also be found on the surface of nanotubular processes that resemble cytonemes and tunneling nanotubes (TNTs), which are actin-filled membrane processes of approximately 200 nm in diameter but often tens of microns in length [28
In , where the cells are co-stained with the red probe, DiIC16(3), and the green cytosolic probe calcein, three enlarged bulb-shaped TVS are observed along a TNT. Red and yellow stained LE/L vesicles are found inside these vesicles, as well as in the interior of the cellular processes, but there is also substantial staining on the cell surface continuous with the plasma membrane of the nanotubular processes that connect the vesicles (). In MLIV cells that express no MLN1 we observed intense intracellular LE/L vesicle staining with this probe, but the plasma membrane was only weakly stained, perhaps due to the deficient trafficking and lysosomal exocytosis in the MLIV cells (). The intensity of the plasma membrane staining in normal and MLIV cells can be compared between , in which the edges of the respective plasma membranes are indicated by open arrows (note the deficiency in PM staining at the edge marked by open arrow on ). In order to quantify the vesicles in each population, we visualized the vesicles under confocal microscopy. , and the enlarged inset shown in 5h, show the typical cross-sections of bulb-shaped vesicle as they appear in single confocal planes (red arrows). Although such vesicles can be observed in MLIV cells (red arrows in ), they tend to be smaller, fewer in number, with lower intensity DiIC16(3) staining (, green arrows in 5i). We used digital image analysis to measure the percentage of total cellular area per field that was occupied by cell surface vesicles (Percent TVS Area). We found that the percentage occupied by vesicles was 64% lower in MLIV fibroblasts than in normal fibroblasts When normalized, the TVS Ratio was 2.77 +/−0.34 fold higher in normal cells, relative to MLIV cells (p <0.01 **; n=14)().
Expression of GPF-tagged MLN1 cDNA constructs in MLIV fibroblasts
To confirm an association between MLN1 and the bulb-shaped TVS on fibroblasts, we transfected MLIV cells with GFP-tagged WT-MLN1 cDNA, or with cDNA constructs with point mutations in the serine lipase (SL and L106P mutants) or ion channel domains (F465L mutant). We verified expression and correct localization of the GFP-tagged fusion protein to the lysosomes. In all constructs, the expressed GFP-tagged protein co-localized with the acidic LysoTracker Red-stained vesicles to a similar extent (). This finding is consistent with the lysosomal localization of MLN1 as shown in other studies [37
]. We were also able to detect translocation of the lysosomes out to the bulb-shaped TVS in WT and mutant cDNA transfections, suggesting that intracellular vesicle trafficking was not affected in the respective mutations (not shown).
Co-localization of MLN1-GFP staining with LysoTracker Red in lysosomes of fibroblasts
We measured the Percent TVS Area in the transfected MLIV cells. We documented the restoration of bulb-shaped TVS on the cell surface of WT-transfected MLIV fibroblasts to levels approaching that of normal untransfected fibroblasts, whereas transfection with pcDNA vector did not significantly alter the level of TVS relative to untransfected MLIV cells. When the data was normalized to the vector-transfected MLIV control, the TVS Ratio of WT-transfected fibroblasts was 2.46 +/−0.37 (p < 0.01 **; n=12), approaching the ratio of 2.77 +/− 0.34 that we found in untransfected fibroblasts ().
Some of the events associated with lysosomal trafficking, including fusion of the lysosomes with the plasma membrane, could potentially be mediated by the opening of the MLN1 channel or the phospholipase activity, or both. The role of the channel in the mechanism of formation of TVS, and its relationship to the serine lipase activity, is not yet understood. For this reason we examined the contribution of the individual domains by expressing MLN1 cDNAs with point mutations that affected either the lipase or channel activity. We expressed two separate cDNAs with lipase point mutants, SL the engineered lipase knockout, and L106P, a naturally occurring point mutation found in an MLIV patient [19
]. We compared the plasma membrane surface topology in the cells transfected with the SL mutant constructs to those transfected with the WT-MLN1 construct or the empty vector. In contrast to the WT-MLN1 construct, neither the SL nor the L106P mutants was able to significantly restore TVS formation on the cell surface above the level of vector transfected MLIV cells, with TVS Ratios of 1.23 +/−0.18 (p=0.28, n=9), and 0.93 +/−0.18 (p=0.73, n=9), respectively (), despite the presence of a functional channel domain in these constructs (). We also transfected the MLIV cells with a cDNA expressing the 465/Phe→Leu mutation (F465L) that inactivates channel function [39
] but contains a normal serine lipase site. It is interesting to note that the F465L-transfected cells showed a significant, albeit limited, restoration of TVS formation (TVS Ratio = 1.68 +/−0.25, (p <0.05 *, n=9)), despite the absence of a functional channel (). These data suggest that both the channel and the serine lipase motif may play separate roles in the formation of TVS, but the serine lipase domain appears to predominate in this aspect of membrane remodeling. The roles of MLN1’s channel and serine lipase domains, as well as the potential contributions of accessory proteins such as TRPML2 and TRPML3 and other endogenous channels may play a partial compensatory role in some of the phases of these processes in MLIV cells, and will need to be examined in separate studies.
Role of MLN1-associated PLA2 activity in membrane remodeling
We have obtained evidence that MLN1 can mediate phospholipase activity by virtue of its serine lipase active site on the large lumenal loop between the first and second transmembrane domains. The results obtained using the three different systems, oocytes, model membrane vesicles and human fibroblasts, corroborate one another, suggesting that the serine lipase active site of MLN1 is required for its effects on membrane remodeling. The TVS in oocytes are larger than those in the model membrane system but most of the TVS are proportionate in size relative to the structures from which they were formed (e.g., oocytes and model membrane vesicles). Further study will be needed to determine whether MLN1 conveys the enzymatic activity directly, or merely facilitates the activity of an associated phospholipase.
The mechanism by which MLN1 influences membrane reshaping remains to be determined. Several potential mechanisms may be considered. Prior studies have demonstrated that direct application of different types of phospholipases, including PLA2, to large liposomes mediated vesicle budding and further remodeling of liposomal membranes [40
]. The effects observed in model membrane systems cannot be explained by activation of signaling pathways and are apparently mediated by direct spontaneous reshaping of the phospholipid bilayers by the phospholipases [43
]. The lysophospholipids produced as cleavage products of PLA2 have inverted conical shapes that promote convex outward orientation of membrane bending, conventionally referred to as positive curvature, on the adjacent leaflet of the bilayer. This has been proposed to influence, in turn, a negative curvature of the other face [44
Potential topological changes related to the roles of MLN1-associated PLA2 activity in membrane remodeling
Role of PLA2-mediated membrane remodeling in the formation of TVS
Considering the localization of MLN1’s lipase domain on the lumenal face of LE/L, it is conceivable that it may act during several potential steps leading to the formation of TVS on the cell surface. We have previously shown that overexpression of MLN1 is associated with enhanced lysosomal exocytosis in human fibroblasts and in Xenopus oocytes coinciding with increased MLN1 channel activity on the plasma membrane, suggesting that MLN1 is translocated to the surface within these patches [1
]. The phospholipase activity associated with the luminal leaflet which would be found on the outer surface of the plasma membrane after exocytosis, is likely to promote the outward curvature. The domain may enable the curvature on the lumenal leaflet at the neck of the fusion junction between the lysosome and the plasma membrane during exocytosis () [42
]. The accumulation of conical lysophospholipids, perhaps in conjunction with the coordinated actions of accessory proteins in physiological systems that promote membrane curvature such as cytoskeletal and scaffold proteins and lysophosphatidic acid acyl transferase (LPAT), may also be involved as the lumenal face is inverted to the extracellular face (6c-e). In addition, the phospholipase may act on the cell surface to modify lipids on the plasma membrane and incorporate them into the growing TVS [48
]. The percentage of inverted cone-shaped lipids in outer vs. inner leaflets could influence the diameter of tubular extensions as well [46
] (). Although the actual role of MLN1 in the formation of TVS remains to be determined, the phospholipase activity associated with the protein may facilitate one or more of these steps in membrane trafficking.
In addition to the membrane reshaping steps proposed above, MLN1 may also be involved in facilitating bilayer fusion as the LE/L membranes are integrated into the plasma membrane. Several recent reports have implicated phospholipases in bilayer fusion both in vitro
and in vivo
]. A number of biophysical studies have been undertaken in recent years to sort out the mechanism by which the phospholipases affect membrane fusion, but it has been suggested that in addition to creating conical lipids that deform the bilayer, lipids may also act by increasing the fluidity within a localized region of the bilayer or generating a defect that creates a local instability in the membrane. The phospholipase activity associated with the integral membrane protein, MLN1, within the double bilayer fusion zone could be directly involved in localized enzymatic degradation of the phospholipids. Thus it could help to overcome the barrier of the double hydrophobic bilayer by creating the local membrane disruption or remodeling needed to initiate the formation of the fusion pore. Local disruptions in either leaflet of the plasma membrane may also serve as initial sites where the growing cytoskeletal structures may mediate or contribute to the formation of membrane protrusions and outgrowth of cellular extensions.
In conclusion, this study shows that MLN1, a channel protein of the TRP family, can mediate phospholipase activity that contributes to the formation of membrane vesicular and tubular structures via its serine lipase active site. Thus MLN1 represents a novel type of bifunctional protein that plays a role in cell surface restructuring and potentially in the formation of cell processes.