P4-ATPase Requirement for AP-1/Clathrin Function in Protein Transport from the trans-Golgi Network and Early Endosomes

Abstract

Drs2p is a resident type 4 P-type ATPase (P4-ATPase) and potential phospholipid translocase of the trans-Golgi network (TGN) where it has been implicated in clathrin function. However, precise protein transport pathways requiring Drs2p and how it contributes to clathrin-coated vesicle budding remain unclear. Here we show a functional codependence between Drs2p and the AP-1 clathrin adaptor in protein sorting at the TGN and early endosomes of Saccharomyces cerevisiae. Genetic criteria indicate that Drs2p and AP-1 operate in the same pathway and that AP-1 requires Drs2p for function. In addition, we show that loss of AP-1 markedly increases Drs2p trafficking to the plasma membrane, but does not perturb retrieval of Drs2p from the early endosome back to the TGN. Thus AP-1 is required at the TGN to sort Drs2p out of the exocytic pathway, presumably for delivery to the early endosome. Moreover, a conditional allele that inactivates Drs2p phospholipid translocase (flippase) activity disrupts its own transport in this AP-1 pathway. Drs2p physically interacts with AP-1; however, AP-1 and clathrin are both recruited normally to the TGN in drs2⌬ cells. These results imply that Drs2p acts independently of coat recruitment to facilitate AP-1/clathrin-coated vesicle budding from the TGN. INTRODUCTIONClathrin mediates protein transport between the plasma membrane, endosomes, and TGN through association with pathway-specific adaptors that link integral membrane cargo proteins to the clathrin lattice during vesicle formation. In mammalian cells, the ubiquitously expressed heterotetrameric AP-1A clathrin adaptor, composed of ␥, ␤1, 1A, and 1-adaptins, appears to mediate both anterograde transport of proteins from the TGN to early endosomes, as well as the retrograde trip back to the TGN (Hinners and Tooze, 2003;Robinson, 2004). For its role in anterograde transport of lysosomal enzymes, AP-1A collaborates with monomeric adaptors called GGAs (Golgi localized, ␥-earcontaining, Arf-binding proteins) to recruit the mannose-6-phosphate receptor into clathrin-coated vesicles (CCVs; Doray et al., 2002). The AP-1B complex, which differs from AP-1A by an alternate 1B subunit, has a functionally distinct role in sorting cargo from the TGN to the basolateral membrane of polarized epithelial cells (Folsch et al., 1999). In budding yeast, there is evidence that AP-1 mediates early endosome to TGN retrograde transport (Valdivia et al., 2002;Foote and Nothwehr, 2006), but no anterograde role for AP-1 has yet been described. The yeast GGAs appear to function independently of AP-1 to mediate delivery of cargo from the TGN to the late endosome (Black and Pelham, 2000). Deletion of genes encoding AP-1 subunits or GGAs has little consequence to growth of yeast. However, the combined deletion of AP-1 and GGA causes a severe growth defect, suggesting that these two adaptors mediate parallel transport pathways and that yeast cannot tolerate loss of both pathways (Costaguta et al., 2001;Hirst et al., 2001).The mechanism of AP-1/clathrin-coated vesicle ...

The Eukaryotic Translation Elongation Factor 1Bγ Has a Non-guanine Nucleotide Exchange Factor Role in Protein Metabolism

“…This provided more evidence that eEF1B␥ plays a role in transport of proteins between the Golgi apparatus and ER. These findings also fit well with the previous identification of eEF1B␥ as a high copy suppressor of cold-sensitive mutations of DRS2, a gene shown to play a role in vesicle budding from the Golgi (12,13), as well as its identification as a calcium-dependent membrane-binding protein (11).…”

“…Significant evidence exists, however, implicating a nontranslation-related role for eEF1B␥. First, eEF1B␥ was found to be a high copy suppressor of cold-sensitive mutations of DRS2, a gene that has been shown to play a role in vesicle budding from the Golgi (12,13). Further, eEF1B␥ was identified in a screen for calcium-dependent membrane-binding proteins (11).…”

“…Yeast strains lacking eEF1B␥ do, however, exhibit significant resistance to oxidative stress in the form of CdSO 4 and H 2 O 2 (10). Additionally, eEF1B␥ has been identified both as a calcium-dependent membrane-binding protein and as a high copy suppressor of a mutation in the DRS2 gene, whose product has been shown to play a role in vesicle budding from the Golgi (11)(12)(13). These findings strongly suggest a role for eEF1B␥ in the membrane systems of the cell, although the exact mechanism by which eEF1B␥ affects this process and the link to gene expression is unknown.…”

A P4-ATPase Protein Interaction Network Reveals a Link between Aminophospholipid Transport and Phosphoinositide Metabolism

“…20,22 While this idea is consistent with the finding that Drs2p physically interacts with Arf activator Gea2p and clathrin adaptor protein AP-1, recruitment of AP-1 and clathrin to the TGN has been shown to occur independently of Drs2p. 23,24 An alternative possibility is that the physical displacement of aminophospholipid from the luminal to the cytosolic leaflet catalyzed by P 4 -ATPases bends the membrane toward the cytosol, and in this way assists the coat machinery during vesicle budding. 20,22 How P 4 -ATPases cooperate with coat proteins in vesicle biogenesis remains to be established.…”

Arl1p regulates spatial membrane organization at the trans -Golgi network through interaction with Arf-GEF Gea2p and flippase Drs2p

“…Therefore, flippases can drive membrane bending toward the cytosol and contribute to vesicle-mediated protein transport by facilitating the generation of membrane curvature that is inherent to this process (23). The inactivation of Drs2p results in defects in the formation of an Arf/ clathrin-dependent class of post-Golgi vesicles that carry exocytic cargo (24). Moreover, a mutation in DRS2 was shown to cause synthetic lethality with clathrin heavy chain and ARF1 (25).…”