Subunit H of the V-ATPase Involved in Endocytosis Shows Homology to β-Adaptins

Abstract

The vacuolar ATPase (V-ATPase) is a multisubunit enzyme that facilitates the acidification of intracellular compartments in eukaryotic cells and plays an important role in receptor-mediated endocytosis, intracellular trafficking processes, and protein degradation. In this study we show that the C-terminal fragment of 350 residues of the regulatory subunit H (V1H) of the V-ATPase shares structural and functional homologies with the ␤-chains of adaptor protein complexes. Moreover, the fragment is similar to a region in the ␤-subunit of COPI coatomer complexes, which suggests the existence of a shared domain in these three different families of proteins. For ␤-adaptins, this fragment binds to cytoplasmic di-leucine-based sorting motifs such as in HIV-1 Nef that mediate endocytic trafficking. Expression of this fragment in cells blocks the internalization of transmembrane proteins, which depend on di-leucine-based motifs, whereas mutation of the consensus sequence GEY only partly diminishes the recognition of the sorting motif. Based on recent structural analysis, our results suggest that the di-leucine-binding domain consists of a HEAT or ARM repeat protein fold. INTRODUCTIONTargeting of transmembrane proteins to different compartments of the endocytic and late secretory pathways depends largely on sorting signals contained within their cytosolic domains (Letourneur and Klausner, 1992;Mellman, 1996;Kirchhausen et al., 1997). These signals are thought to interact with specific recognition molecules, which are components of membrane-bound transport intermediates (Schmid, 1997;Hirst and Robinson, 1998;Bonifacino and Dell'Angelica, 1999;Kirchhausen, 1999;Marsh and McMahon, 1999). Most internalized proteins contain sorting signals such as the tyrosine-based motif, Yxx , where x is any amino acid and is a bulky hydrophobic side chain (Lobel et al., 1989;Collawn et al., 1990;Boll et al., 1996) or the di-leucine-based motif, LL (Letourneur and Klausner, 1992). For the tyrosine-based motif, the interaction is mediated by the medium chains of adaptor protein (AP) complexes (Ohno et al., 1995). Cocrystallization of the signal binding domain of 2 with two different Yxx peptides provided a structural explanation for this binding specificity (Owen and Evans, 1998).The Nef protein of primate lentiviruses is the only nontransmembrane protein known to traffic via a di-leucinebased motif (Kirchhausen, 1999). It is required for the internalization of CD4 from the cell surface to endosomes and lysosomes (Craig et al., 1998;Greenberg et al., 1998). Several proteins have been proposed to direct the sorting of Nef. They include the ␤-chain of AP-1 complexes (Bresnahan et al., 1998;Greenberg et al., 1998), the ␤-subunit of the COP-I coatomer (Piguet et al., 1999;Janvier et al., 2001), and the regulatory subunit H (V1H) of the vacuolar ATPase (VATPase), previously named NBP1 for Nef binding protein 1 (Lu et al., 1998;Mandic et al., 2001). Although the precise sites of these interactions varied, all protein complexes were reported to bind ...

Protein homeostasis of a metastable subproteome associated with Alzheimer’s disease

“…Hence, RAB6A, which is a central gene in the AD metastable network described in this work, could play an important role in the regulation of the metastable proteins by directing them toward the endosomallysosomal degradation machinery, thereby preventing their accumulation in the cytoplasm. Another two genes in the group that we found are ATP6V1H, which encodes a protein subunit of a vacuolar ATPase involved in clathrin-mediated endocytosis (57,58) and whose role in regulating lysosomal pH has been recently been linked to neurodegeneration (59), and ATL1, which is involved in ER trafficking (60,61). In fact, all 10 genes that we found to be related to trafficking are part of the endosomal-lysosomal system.…”

Renal Vacuolar H+-ATPase

“…The carboxy-terminal portion of subunit H shares significant homology with adaptor protein complexes and is similar to the ␤-subunit of COPI coatomer complexes (196). This subunit binds to the carboxy-terminal loop of Nef and to the adaptor protein complex 2 (AP-2) (197).…”

Human Immunodeficiency Virus Type 1 Nef: Adapting to Intracellular Trafficking Pathways

“…Several models for Nef-mediated CD4 transport exist, each based on a familiar premise: Nef binds to the cytoplasmic tail of CD4 and recruits a cellular factor(s) to transport CD4 from the cell surface to lysosomes for degradation. Several potential cellular factors have been proposed, based on the correlative data that mutations that effect their binding are also necessary for Nef activity: V1H binding requires the dileucine and diaspartic acid motifs (56,59,102), AP-2 binding requires the dileucine motif (37), and both ARF1 binding and ␤-COP binding require the diglutamic acid motif (52, 123) (although not all investigators have observed this dependency [77]). …”