Báo cáo khoa học: Vps4 regulates a subset of protein interactions at the multivesicular endosome

Vps4 Regulates a Subset of Protein Interactions at the Multivesicular Endosome

Tác giả: Parimala R. Vajjhala, Elizabeth Catchpoole, Chau H. Nguyen, Carol Kistler và Alan L. Munn

Lĩnh vực: Sinh học phân tử, Khoa học sinh học y tế

Nội dung tài liệu:
Nghiên cứu này tập trung vào vai trò của Vps4 trong việc điều chỉnh các tương tác protein tại thể đa nang ngoại bào (multivesicular endosome – MVB). MVB đóng vai trò quan trọng trong nhiều quá trình sinh học, bao gồm điều hòa thụ thể, trình diện kháng nguyên và tín hiệu giao tiếp ngoại bào qua exosome. Máy móc phân tử của MVB cũng được sử dụng trong quá trình nảy chồi của virus. Vps4, một enzyme AAA ATPase, được cho là có vai trò thiết yếu trong việc phân rã các thành phần của máy móc MVB.
Nghiên cứu này đã phân tích một cách có hệ thống các tương tác của Vps4 với các thành phần khác của máy móc MVB. Kết quả cho thấy Vps4 tương tác trực tiếp với Vps2 và Bro1. Một tập hợp các tương tác của Vps4 được điều hòa bởi quá trình thủy phân ATP, và một tương tác được điều hòa bởi sự gắn kết ATP. Đáng chú ý, phần lớn các protein tương tác với Vps4 thực hiện thông qua miền N-terminal MIT của nó. Các phát hiện này cho thấy miền MIT có vai trò kép trong việc gắn kết cơ chất và tuyển dụng đến thể ngoại bào, và Vps4 phân rã máy móc MVB bằng cách tác động trực tiếp lên nhiều protein.

Mục lục chi tiết:

• Keywords: endocytosis; lysosome; macromolecular disassembly; membrane traffic; vacuole
• Correspondence
• Abbreviations
• During endocytic transport, specific integral membrane proteins are sorted into intraluminal vesicles that bud from the limiting membrane of the endosome. This process, known as multivesicular body (MVB) sorting, is important for several important biological processes. Moreover, components of the MVB sorting machinery are implicated in virus budding. During MVB sorting, a cargo protein recruits components of the MVB sorting machinery from cytoplasmic pools and these sequentially assemble on the endosome. Disassembly of these proteins and recycling into the cytoplasm is critical for MVB sorting. Vacuolar protein sorting 4 (Vps4) is an AAA (ATPase associated with a variety of cellular activities) ATPase which has been proposed to play a critical role in disassembly of the MVB sorting machinery. However, the mechanism by which it disassembles the complex is not clear. Vps4 contains an N-terminal microtubule interacting and trafficking (MIT) domain, which has previously been shown to be required for recruitment to endosomes, and a single AAA ATPase domain, the activity of which is required for Vps4 function. In this study we have systematically characterized the interaction of Vps4 with other components of the MVB sorting machinery. We demonstrate that Vps4 interacts directly with Vps2 and Brol. We also show that a subset of Vps4 interactions is regulated by ATP hydrolysis, and one interaction is regulated by ATP binding. Finally, we show that most proteins interact with the Vps4 MIT domain. Our studies indicate that the MIT domain has a dual role in substrate binding and recruitment to endosomes and indicate that Vps4 disassembles the MVB sorting machinery by direct effects on multiple proteins.
• During endocytic trafficking, some integral membrane proteins are sorted into internal vesicles which form by invagination of the endosome limiting membrane. This process, referred to as multivesicular body (MVB) sorting, is critical for a number of important biological processes including receptor down-regulation, antigen presentation and exosome-dependent intercellular signalling (reviewed in [1–3]). Interest in the mechanism of MVB sorting has escalated since the discovery that components of the MVB sorting machinery are also utilized for virus budding, a process topologically similar to MVB sorting (reviewed in [4,5]).
• The process of MVB sorting was first examined in mammalian cells [6,7], but the components of the MVB sorting machinery were first characterized in Saccharomyces cerevisiae [8–10]. The pathway is highly conserved from yeast to mammalian cells, although the number of components is expanded in mammalian cells because of the multiplicity of isoforms [11]. Recognition of a cargo protein, usually by the presence of a ubiquitin molecule, is followed by sequential recruitment of components of the MVB sorting machinery. The order of recruitment of the different components to the endosome membrane is starting to emerge, and structural data have recently been obtained for several components (reviewed in [12]). A critical process during MVB sorting is the disassembly of the MVB sorting machinery, which allows recycling and new rounds of vesicle budding. However, the molecular mechanisms that regulate cycling of the MVB sorting machinery on and off endosomes is not yet well understood.
• Vps4p, also known as Csclp, End13p, Grd13p, Vpl4p, Vpt10p, or Did6p, is the only essential component of the MVB sorting machinery with known enzymatic activity. It is a member of the AAA (ATPase associated with a variety of cellular activities) family of ATPases [13,14]. Mammalian cells express two isoforms of VPS4, VPS4A and VPS4B, and both proteins function in endocytic trafficking [15–17]. All members of the AAA superfamily contain one or two copies of a conserved ATPase domain (AAA module). Although not known for Vps4p, other AAA ATPases assemble into oligomeric rings. Distinct members of the AAA ATPase family function in diverse cellular processes, but a common theme is protein unfolding and macromolecular disassembly (reviewed in [18,19]).
• Loss of Vps4p function in yeast and mammalian cells disrupts MVB sorting and results in the formation of an aberrant multilamellar endosomal compartment referred to as the class E compartment [8,14]. As both endocytic and biosynthetic traffic to the lysosome/vacuole proceeds via the MVB, the class E compartment accumulates endocytic and biosynthetic material as well as late Golgi proteins because of defective trafficking out of this compartment [9]. In yeast, defective recycling of late Golgi proteins including the receptor that transports soluble vacuolar proteins from the Golgi to the MVB results in missorting and secretion of soluble vacuolar proteins to the extracellular medium [20,21].
• Loss of Vps4p function is also accompanied by the redistribution of several components of the MVB sorting machinery from the cytoplasm to endosomes [22,23]. Therefore Vps4p-dependent ATPase activity has been proposed to be important for disassembly of the MVB sorting machinery and release into the cytoplasm. An N-terminal microtubule interacting and trafficking (MIT) domain is required for recruitment of Vps4p to endosomes [22,24,25], but it is not clear precisely how Vps4p disassembles the MVB sorting machinery. Previous work from our laboratory has shown that Vps20p, a component of the MVB sorting machinery, interacts with Vps4p and dissociates from Vps4p upon ATP hydrolysis [26]. This was the first evidence that Vps4p ATPase activity can break intermolecular interactions. As Vps20p is a coiled-coil protein and interacts with the N-terminal MIT domain of Vps4p, Vps4p ATPase activity may break coiled-coil interactions and thereby disassemble the MVB sorting machinery.
• Several putative interactions have been reported between Vps4p and components of the MVB sorting machinery [27-30]. However, as there is a complex network of interactions between the components of the MVB sorting machinery, it was not clear which interactions with Vps4p are direct and which are indirect. In addition, it was not clear how many of these putative Vps4p interactions with the MVB sorting machinery may be regulated by Vps4p ATPase activity. That not all Vps4p interactions are regulated by ATPase activity is supported by our previous studies showing that the Vps4p interaction with Vta1p is not affected by ATP hydrolysis [26].
• Here, we demonstrate new direct interactions between Vps4p and the MVB sorting machinery in yeast. We present evidence that a subset of Vps4p interactions is regulated by ATP hydrolysis, and that one interaction is regulated by ATP binding to Vps4p. Finally, we also show that most Vps4p interactors interact with the MIT domain of Vps4p.
• Results
• Vps4p binds directly to multiple components of the MVB sorting machinery
• Vps4p has been shown to bind directly to a few MVB sorting machinery components including Vps20p, Vta1p and Did2p/Chm1p [26,31]. However, it is not known whether the function of Vps4p in disassembly of the MVB sorting machinery is mediated solely via interactions with these proteins or whether Vps4p binds directly to and acts via other components of the MVB sorting machinery. We therefore tested candidate Vps4p interactions with components of the MVB sorting machinery. When binding of purified Vps4p to MVB components was examined in vitro, Vps4p was found to bind directly to multiple MVB sorting machinery components, including Vps2p and Bro1p, in addition to Vps20p, Vta1p and Did2p, but not to Snf7p (Fig. 1). Our data also show that, when molar equivalent amounts of Vps4p interactors are compared, the amount of Vps4p that binds to Did2p is greater than that bound by any of the other interactors. These data are consistent with Vps4p having a relatively high affinity for Did2p.
• ATP binding and ATP hydrolysis by Vps4p regulates protein interactions at the MVB
• Vps4p has been proposed to function as a protein complex disaggregation machine on endosomes [22]. Consistent with this, the binding of Vps20p to Vps4p is regulated by ATP hydrolysis [26]. It was likely that other components of the MVB sorting machinery are also substrates for disassembly by Vps4p. To test this, we performed the in vitro binding assay in the presence and absence of ATP. In the presence of ATP, purified wild-type Vps4 is catalytically active and will hydrolyse added ATP. Thus interactions that are regulated by Vps4p ATPase activity are predicted to decrease under these conditions. In contrast, interactions that are not regulated by Vps4p ATPase activity are predicted to be unaffected. The data obtained show that binding of both Vps2p and Bro1p to Vps4p was decreased in the presence of ATP compared with binding in the absence of ATP. However, the binding of Did2p to Vps4p was not affected by the presence of ATP (Fig. 2).
• To determine whether the decreased binding in the presence of ATP is due to ATP hydrolysis or ATP binding, the effect of ATP on binding to a Vps4p mutant (Vps4p-E233Q) that is defective in ATP hydrolysis was also studied. Binding of Vps2p and Vps20p to Vps4p-E233Q was increased in the presence of ATP, but binding of Bro1p to Vps4p-E233Q was decreased in the presence of ATP (Fig. 2).
• We surmise that the decreased binding of Vps2p to Vps4p in the presence of ATP is due to Vps4p-dependent ATP hydrolysis. In contrast, the decreased binding of Bro1p to wild-type Vps4p and to Vps4p-E233Q in the presence of ATP may be due to competitive binding or an allosteric effect.
• Several components of the MVB sorting machinery interact with Vps4p via the N-terminal MIT domain
• To determine whether there is any correlation between the binding sites on Vps4p and the response of the interacting proteins to ATP binding and hydrolysis, we determined the region of Vps4p that mediates interaction with Vps2p, Snf7p and Bro1p using the yeast two-hybrid technique (Fig. 3). Both Vps2p and Snf7p interact with Vps4p mainly via the Vps4p N-terminal coiled-coil domain (Fig. 3C). We did not detect an interaction between Bro1p and full-length Vps4p or any of the Vps4p domains using the yeast two-hybrid technique (not shown) consistent with a previous report [27].
• To more precisely map the interaction sites of Vps2p, Snf7p, Vps20p, and Did2p within the Vps4p N-terminal domain, we generated two Vps4p N-terminal mutants (Fig. 4A,B) and tested the effect of these mutations on the different Vps4p interactions. In the Vps4p-YEE mutant, residues 26–29 in the second helix of the MIT domain were deleted. These residues are completely conserved between the yeast and human VPS4 isoforms. In the Vps4p-IRA mutant, residues 56–71 in the third helix of the MIT domain were deleted. These residues are also highly conserved. The YEE and IRA mutants are named after the first three amino acids that were deleted in each motif. We also tested the effect of a previously described Vps4p-coiled-coil (CC) mutation [22] in which residues 50–87, which comprises most of the second and third helices of the MIT domain, were deleted.
• Yeast two-hybrid analysis (Fig. 4C) revealed that each mutation diminished but did not abolish Did2p interaction, whereas Vps2p interaction was abolished by the Vps4p-IRA and Vps4p-CC mutations. All the N-terminal mutations tested abolished Vps20p and Snf7p interactions. As expected, the interaction of Vta1p with the Vps4p C-terminal domain was not diminished by any of the N-terminal mutants we tested.
• To confirm the yeast two-hybrid interactions described above and to identify the domain of Vps4p to which Bro1p binds, we performed in vitro protein-binding assays (Fig. 4D). The data obtained indicate that the Vps4p-YEE and Vps4p-IRA mutations diminish direct binding of Vps2p, Vps20p and Did2p. The Vps4p-CC mutation appeared to increase binding to all interactors. However, the Vps4p-CC mutant protein also displayed an interaction with glutathione S-transferase (GST) alone (Fig. 4D) and displayed an increased interaction with the Vps4p antibody (not shown). Thus, we cannot interpret the data obtained for this Vps4p-CC mutant protein. Mutation of the β domain abolished interaction with Vta1p as previously reported [32,33] and in addition increased binding to the N-terminal interactors, including Vps2p, Vps20p and Did2p. None of the N-terminal MIT domain mutations or the C-terminal β domain mutations diminished binding of Vps4p to Bro1p.
• In summary, our data indicate that most Vps4p-interacting proteins (Vps2p, Vps20p, Did2p and Snf7p) interact with the N-terminal MIT domain of Vps4p. In addition, these data show that the Bro1p interaction with Vps4p is unique as it is undiminished by mutations in the N-terminal MIT domain or β domain.
• Vps4p interactions with Did2p, Vps2p and Vps20p are important for recruitment to endosomes and for MVB sorting
• To determine whether the Vps4p YEE and IRA motifs play a role in Vps4p recruitment to endosomes, wild-type and mutant Vps4p tagged with green fluorescent protein (GFP) were expressed in cells in which the chromosomal VPS4 gene has been deleted (vps4Δ). Wild-type GFP-tagged Vps4p could be detected on punctate structures (Fig. 5A) consistent with localization to endosomes, as previously reported [22]. However, the GFP-tagged Vps4p-YEE and Vps4p-IRA mutant proteins, like the GFP-tagged Vps4p-CC mutant protein, exhibited severely reduced punctate localization. We conclude that the Vps4p YEE and IRA motifs are important for Vps4p recruitment to endosomes.
• To test whether the YEE and IRA motifs and the region deleted in the Vps4p-CC mutant protein are important for Vps4p function in vivo, we tested the ability of the N-terminal mutant proteins to restore peptidase Y compared with vps4Δ cells transformed with empty vector alone (Fig. 5C). We conclude that the YEE and IRA motifs as well as the region deleted in the Vps4p-CC mutant are important for Vps4p function in MVB sorting and vacuolar protein sorting.
• To test whether the phenotypes of vps4Δ cells expressing the Vps4p N-terminal mutants were due to lowered expression or degradation of the mutant proteins, we tested the steady-state expression of the mutant Vps4p proteins (Fig. 5D). Although both the Vps4p-IRA and Vps4p-YEE mutants are expressed, their steady-state connections are somewhat reduced compared with that of wild-type Vps4p. However, such a modest reduction in expression level is unlikely to account for the inability of these mutant proteins to restore MVB sorting and vacuolar protein sorting in vps4Δ yeast. Surprisingly, the expression of the Vps4p-CC mutant was significantly greater than that of wild-type Vps4p. We conclude that the phenotypes observed in vps4Δ cells expressing the Vps4p N-terminal mutants are due to loss of function of these mutant proteins.
• We surmise that the interactions of the Vps4p MIT domain with Did2p, Vps2p, Vps20p and Snf7p are critical for Vps4p recruitment to endosomes and Vps4p function in MVB sorting.
• Discussion