An increasing body of evidence suggests a tight connection between inositol phospholipids and sphingolipid metabolism. In Saccharomyces cerevisiae
, one of the PI4Ks, Stt4p, is linked to the regulation of sphingolipid biosynthesis (Tabuchi et al., 2006
), as is Sac1p (Brice et al., 2009
; Breslow et al., 2010
), the phosphatase that regulates PtdIns4P
levels both in the ER and the plasma membrane (Foti et al., 2001
; Stefan et al., 2011
). In mammalian cells, several lipid transport proteins contain PtdIns4P
-binding PH domains, including the ceramide transfer protein CERT (Hanada et al., 2003
) and the GlcCer transfer protein FAPP-2 (D'Angelo et al., 2007
production is therefore critical to the regulation of sphingolipid biosynthetic pathways, ensuring that Cer and GlcCer reach the appropriate Golgi compartment for synthesis of sphingomyelin and complex glycosphingolipids, respectively. Remarkably, the PI4K enzyme that produces PtdIns4P
differs for the two processes: PI4KIIIβ is primarily responsible for CERT (Toth et al., 2006
), whereas PI4KIIα is more important for FAPP-2 function (D'Angelo et al., 2007
). The present study extends our understanding of the regulation by these PI4Ks to the catabolism of sphingolipids by revealing a concerted role of the two kinases in the trafficking and delivery of one of the key enzymes of sphingolipid breakdown to the lysosome.
Our analysis has clearly shown that PtdIns4P
, especially the pool made by PI4KIIIβ, plays an important role in exit of LIMP-2 from the Golgi. This conclusion was reached by several observations using a variety of approaches. First, either acute elimination of PtdIns4P
from the Golgi by a recruited Sac1 phosphatase or inhibition of PI4KIIIβ with a selective inhibitor, PIK93, resulted in the retention of LIMP-2-GFP in the Golgi. Second, PIK93 treatment prevented exit of a LIMP-2-PA-GFP construct from the Golgi compartment. Third, PIK93 also inhibited the missorting of the GBA enzyme into the medium under conditions that prevented GBA reaching the lysosomes. An important observation of this series of experiments was the selective effect of PI4KIIIβ inhibition by PIK93 on LIMP-2-GFP but not on CI-M6PR, another receptor that is responsible for lysosomal delivery of several hydrolases. This finding is in agreement with earlier reports that CI-M6PR, a bona fide AP-1 cargo, requires PtdIns4P
synthesized by PI4KIIα for proper trafficking along the degradative pathway (Wang et al., 2003
) and that total elimination of PtdIns4P
also prevented exit of this receptor from the Golgi (Szentpetery et al., 2010
). This critical observation was further underlined by a clear segregation within the Golgi of the CI-M6PR and an mRFP-tagged LIMP-2 tail after PIK93 treatment. Together these data suggest the existence of Golgi membrane subdomains, in which PtdIns4P
made by distinct PI4Ks controls the exit of select cargo molecules.
Owing to the lack of inhibitors for type II PI4Ks, the role of this enzyme was studied by RNAi-mediated knockdown, which alters trafficking pathways on a different timescale. Nevertheless, PI4KIIα knockdown cells displayed a very characteristic phenotype, namely accumulation of LIMP-2-GFP molecules in large, closely aggregated vesicles that are likely prelysosomal sorting intermediates. This effect was reversed either by a catalytically active or an inactive PI4KIIα, but not by the AP-3–binding LL mutant. Interestingly, both kinase-dead and AP-3 binding–deficient mutants failed to significantly rescue the GBA secretion defect induced by PI4KIIα knockdown, indicating the apparent rescue of the morphological defect by this variant does not reflect full recovery of the segregation between endocytic/lysosomal compartments and correct acidification. Since PI4KIIα KD still exhibits ~50% of AP-3 binding (Craige et al., 2008
), this explains the partial ability of PI4KIIα KD to alleviate the morphological defect. A similar observation was made recently by Craige et al
., who showed enlarged prelysosomal vesicles containing LAMP-1 after PI4KIIα knockdown. However, unlike LIMP-2, this LAMP-1 phenotype was partially reversed only by a catalytically active enzyme, but not by an inactive kinase. These authors also showed that association of the PI4KIIα via a dileucine motif with AP-3 was important for supporting the role of the kinase in maturation of LE (Craige et al., 2008
). The connection between AP-3 and LIMP-2 has already been revealed in several studies showing selective binding of the dileucine-based sorting motif of LIMP-2 to AP-3 (Honing et al., 1998
; Le Borgne et al., 1998
; Janvier et al., 2003
An intriguing observation in the course of our studies was revealed by a comparison of the distribution of LIMP-2 and LAMP-1 in cells depleted in PI4KIIα. Remarkably, while the enlarged vesicles entrapped both of these proteins, in most of these enlarged vesicles the two molecules were clearly segregated into distinct subdomains (see ). This observation suggested a lateral separation of these molecules and different processing during their normal trafficking through these compartments. Indeed, unlike LIMP-2, most LAMP-1 is first transported to the plasma membrane and endocytosed prior to reaching lysosomes (Janvier and Bonifacino, 2005
). Also, during maturation of LE, LAMP-1 preferentially clusters within cholesterol-rich domains of the growing limiting membrane (Du et al., 2011
). Depletion of PI4KIIα, therefore, attenuates plasma membrane-to-lysosome trafficking of LAMP-1, as well as Golgi-to-lysosome LIMP-2 transport, resulting in accumulation of the two molecules at an enlarged common compartment. Importantly, LIMP-2 accumulation in the partially acidic enlarged endosomes leads to a dramatic increase in GBA secretion into the medium under PI4KIIα knockdown. Consistent with the role of PI4KIIIβ in LIMP-2 exit from the Golgi, inhibition of PI4KIIIβ in the presence of PI4KIIα RNAi prevented this increased GBA secretion.
Impaired sorting of cargoes to lysosomes after PI4KIIα knockdown has already been observed by Minogue and colleagues, who reported that endocytosed EGF receptors were not efficiently degraded in cells depleted in PI4KIIα (Minogue et al., 2006
). An important question, however, is whether the lysosomal sorting defects are caused by altered sphingolipid metabolism, since inhibition of sphingolipid synthesis also impacts trafficking of other enzymes to lysosome-related organelles. For example, in melanocytes derived from GlcCer synthase knockout mice, delivery of tyrosinase to melanosomes is impaired, resulting in Golgi accumulation of the enzyme and loss of pigmentation (Sprong et al., 2001
). M6PR-mediated lysosomal transport in these cells, however, remains unaltered, consistent with a specific role for glycosphingolipid regulation of M6PR-independent trafficking pathways, including LIMP-2-mediated GBA transport. Similarly, loss of GlcCer synthase led to altered sorting of LAMP-1 to lysosomes, inducing a switch to an alternate, intracellular trafficking route, rather than LAMP-1 traversing via the plasma membrane (Groux-Degroote et al., 2008
). Since PI4KIIα regulates GlcCer transport and formation of complex glycosphingolipids (D'Angelo et al., 2007
), and even appears to play a role in the enhanced sphingomyelin synthesis induced by 25-oxysterols (Banerji et al., 2010
), it remains to be determined whether the blockade of LIMP-2/GBA delivery to lysosomes is a direct effect in the maturation and fusion of these compartments due to lack of the kinase or an indirect consequence of altered sphingolipid synthesis.
The question then arises how PtdIns4P
can regulate the trafficking of LIMP-2 and, even more importantly, how the same lipid made by two different enzymes can regulate the transport of the molecule through different compartments, namely the Golgi and LE. There is an increasing body of evidence that phosphoinositides act in concert with small GTP-binding proteins and clathrin adaptors to regulate cargo selection (Brown et al., 2001
; Christoforidis et al., 1999
; Wang et al., 2003
). Therefore it is conceivable that PtdIns4P
made at the Golgi by PI4KIIIβ regulates the formation of a protein complex that drives the exit of LIMP-2 from the Golgi, while PI4KIIα at the TGN is important at a subsequent stage, helping to assemble a signaling domain that interacts with LIMP-2 and directs it to the LE/lysosome. The exact proteins involved in these different stages of transport must be identified in future studies, but it is likely that AP-3 is an important component at the sorting step regulated by PI4KIIα (Craige et al., 2008
; Salazar et al., 2009
It is important to note that neither of the PI4Ks showed a particularly prominent colocalization with LIMP-2 (or GBA) in COS-7 cells. The differential steady-state localization of PI4KIIIβ at the Golgi and LIMP-2 primarily in lysosomes suggests an interaction that may be confined to a transition state only affecting a small fraction of the respective proteins at any given time. This may explain our inability to find these proteins in complex using immunoprecipitation coupled with Western blotting. Nonetheless, the more sensitive proteomic analysis was able to reveal the association of a small fraction of the LIMP-2 and GBA proteins with PI4KIIIβ that remains below the detection limit of Western analysis following immunoprecipitation. It will be important to determine in future studies whether PI4KIIIβ directly interacts with LIMP-2/GBA or via other protein intermediates.
In summary, the present studies demonstrate a sequential role of two distinct PI4Ks in the lysosomal delivery of the GBA enzyme via its receptor, LIMP-2 (). PI4KIIIβ has a major role in exit of LIMP-2 from the Golgi, whereas PI4KIIα has a role supporting trafficking steps between the LE and lysosomes. These results are the first to implicate PtdIns4P
and PI4Ks in the regulation of the catabolism of complex sphingolipids, adding new details to the biology of the GBA enzyme, a protein known for its prominent role in Gaucher disease. The central role of PI4Ks in integrating the synthetic and degradative pathways of sphingolipids in mammalian cells suggests an evolutionarily conserved connection between phosphoinositides and sphingolipids from yeast to humans. The role of these lipid kinases in the controlling lysosomal function may explain the late onset spinocerebellar degeneration observed in PI4KIIα gene-trap mice (Simons et al., 2009
) and raises the possibility that impaired PI4K functions may contribute to other neurodegenerative disorders.
FIGURE 6: Model of PI4K regulation of LIMP-2-mediated GBA transport. Binding of GBA to LIMP-2 in the ER lumen results in trafficking of this complex to the Golgi. Efficient exit out of this compartment then depends on availability of PtdIns4P and the catalytic (more ...)