Batten disease or JNCL is a severe neurodegenerative disorder characterized by the accumulation of autofluorescent material in the lysosomes of cells derived from afflicted individuals. This phenotype implies defects in the sorting of material away from the lysosome or the faulty degradation of normally trafficked materials or both. In the course of our work, we have found that a Batten disease-related protein in yeast, Btn2 (18
), acts upon protein trafficking from late endosomes. In particular, cells lacking BTN2
accumulate a fluorescent protein-tagged Golgi marker (Yif1) in the vacuole (Fig. ; Table ), as shown earlier (14
), where it undergoes degradation (Fig. ). Yif1 mislocalization also occurs in mutants preferentially defective in late endosome-Golgi trafficking (Fig. ; Table ); thus, Btn2 is likely to act upon this same transport step. Defects in late endosome-Golgi protein recycling may, therefore, be the mechanism underlying Batten disease/NCL pathogenesis in mammals.
encodes a Hook1 ortholog (Fig. ) that is a late component of the endocytic pathway (Fig. ) and which localizes adjacent to the vacuole but is clearly nonnuclear (Fig. ). Btn2, along with the Rhb1 small GTPase, appears to play a prominent role in the recycling of Yif1, as the deletion of either gene results in Yif1 missorting to the vacuole (Fig. ; Table ). Thus, we propose that both Btn2 and Rhb1 mediate late endosome-Golgi recycling. This is supported by several lines of evidence. First, known late endosome-Golgi trafficking mutants, such as the vps26Δ
strain, have a similar effect upon Yif1 sorting (Fig. ; Table ), and GST-Vps26 binds directly to His6
-Btn2 in in vitro binding assays (Fig. ). VPS26
encodes a component of retromer, a pentameric complex composed of Vps5, Vps17, Vps26, Vps29, and Vps35 (73
). Retromer regulates the trafficking of the CPY receptor, Vps10, and retrieves it from the late endosome to the Golgi apparatus. Vps26 has been proposed to link both cargo selection and retromer assembly, while other retromer components, Vps5 and Vps17, form a membrane-associated subcomplex and are the yeast equivalents of sorting nexins 1 and 2 (Snx1 and -2) (73
). These and other retromer components have all been shown to localize to the late endosome (73
). Second, Btn2 localizes to large perivacuolar structures that are consistent with late endosomes (Fig. and ). Btn2 colocalizes in part with the sorting nexin Snx4, the Vps27 late endosome/MVB marker, and the Tlg2 endosomal t-SNARE to these same structures located adjacent to the vacuole (Fig. ). Third, Btn2 coimmunoprecipitates with Yif1, Snx4, and retromer (Fig. ), along with an endosomal SNARE complex (Fig. ), suggesting that it may be a component of an assembled retromer-mediated Yif1 sorting complex. Finally, in vitro binding studies (Fig. ) show that recombinant Btn2 binds to specific components of this putative complex, including the recycling v-SNARE, Snc1, which recycles from early endosomes to the Golgi apparatus (47
) and, by inference from this work, from late endosomes to the Golgi apparatus. In addition, recombinant Btn2 binds to Vps26, which is involved in retromer assembly and cargo sorting (73
). Thus, we propose that Btn2 confers cargo retrieval (i.e., Yif1 and, possibly, SNAREs) from late endosomes to the Golgi apparatus along with retromer.
Interestingly, neither CPY nor Vps10 trafficking was affected by the deletion of BTN2
(Fig. ), although both should access the late endosome on their way to the vacuole in a retromer-dependent fashion (19
). This may imply that there are multiple retromer-mediated cargo sorting events occurring at the late endosome or perhaps that there are multiple late endosomal compartments serviced by retromer (7
). In either case, some may be mediated by Btn2 and others may not, although at this point we cannot distinguish between these two distinct possibilities. Despite this, we were able to show that Btn2 forms complexes with Yif1, retromer, and Snx4 but not with Vps10 (Fig. and data not shown). This is consistent with the idea that there are distinct nexin-retromer sorting complexes operating in yeast. That neither Snc1 recycling from early endosomes to the Golgi apparatus (Fig. ) nor protein export to the plasma membrane (as assayed by calcofluor sensitivity and growth assays [data not shown]) are affected in btn2Δ
cells suggests that the role of Btn2 is limited to regulating the trafficking of specific cargo molecules from a late endosome to the Golgi apparatus. This is supported by the other physical interaction, colocalization, and protein trafficking data presented here. Models outlining the trafficking functions mediated by Btn2 in wild-type cells and the consequences of BTN2
disruption therein are shown (Fig. ).
FIG. 9. A model for Btn2 function in late endosome-Golgi protein sorting. Wild-type cells: the Snc1 exo/endocytic v-SNARE (blue) and Fur4 (red) are both endocytosed and delivered by endocytic vesicles to the early endosome (EE). Snc1 is delivered to the trans (more ...)
In this study, we found that Btn2 interacts physically and genetically with SNAREs (Fig. and ) involved in endocytosis and endosomal sorting (e.g., Snc, Tlg1, Tlg2, and Vti1) (2
). In vitro binding studies reveal a direct interaction with Snc1, which may help recruit Btn2 to the endosomal SNARE complex. This may facilitate interactions with Yif1 (14
) (Fig. ) and retromer (Fig. ) and might be dependent on the Rhb1 small GTPase, which is known to interact with Btn2 (13
) and whose deletion phenocopies btn2Δ
cells (Fig. ). More work is clearly necessary to determine the temporal order of events leading to the assembly of the putative retrieval complex in which Btn2 functions, along with retromer, Snx4, and SNAREs. Finally, there is a connection to COPI which bears future exploration. First, systematic interaction studies have demonstrated that Sec27, a COPI component, and Btn2 interact physically (39
). Second, a role for COPI in late endosome-MVB sorting events has been described for mammalian cells (3
). Third, we recently demonstrated a potential role for specific COPI subunits (COPI B) (52
), including Sec27, in the delivery of CPS, Ste2, Ste3, and Fur4 to multivesicular bodies in yeast (27
). Fourth, retromer components cosediment with COPI-coated membranes when separated by size exclusion chromatography (74
). Finally, work in progress suggests that Btn2 interacts genetically and physically with COPI B components (R. Kama and J. E. Gerst, unpublished results). Thus, Btn2 might function in conjunction with COPI, as well as with retromer, in mediating specific post-Golgi trafficking events.
Previous studies have shown that Btn2 interacts with a wide variety of proteins (i.e., Rhb1, Yif1, and Ist2) (13
) purportedly involved in numerous processes relevant to cell growth and homeostasis (i.e., ion, pH, or amino acid balance). However, the precise role of Btn2 in intracellular membrane transport has remained obscure. Here we reveal that role to be the regulation of cargo protein recycling from the late endosome. Thus, we predict that changes in ion, pH, or amino acid homeostasis, seen in the absence of Btn2, result from the improper recycling of cargo proteins within the endomembrane transport system. If so, this would preclude a general role in the regulation of cell homeostasis, as predicted by Kim et al. (43
). Furthermore, we predict that Batten disease/JNCL in humans may originate from the failed recycling of cargo proteins to their proper sites of action. This might result in the accumulation of nonrecycled cargo in other intracellular compartments (i.e., lysosome) and lead to cellular dysfunction. Interestingly, a connection between the defects in retromer function and the production of the amyloid-β peptide, which is involved in the pathogenesis of Alzheimer's disease, has been noted (76
). Along with this study, it suggests that alterations in late endosome-Golgi protein recycling may play a significant role in the control of cell metabolism and viability.
Finally, an analysis of a role for Btn1 in endomembrane trafficking would seem to be a high priority, in light of the results obtained with Btn2. BTN1
encodes the yeast ortholog of CLN3
), a gene known to be mutated in NCL patients (30
). Btn1 has been proposed to play a role in vacuolar pH homeostasis (57
) and the import of basic amino acids therein (44
), although the mechanism is unknown. Our results may suggest a potential role for Btn1/Cln3 in endosomal protein sorting, which could account for its effects upon vacuolar/lysosomal homeostasis.