Role for Drs2p, a P-Type Atpase and Potential Aminophospholipid Translocase, in Yeast Late Golgi Function

Chen, Chih‐Ying; Ingram, Michael F.; Rosal, Peter H.; Graham, Todd R.

doi:10.1083/jcb.147.6.1223

Cited by 245 publications

(354 citation statements)

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“…Other data linking Drs2p to clathrin function include their colocalization to the TGN, a strong synthetic lethal relationship between drs2, arf1, and chc1 (clathrin heavy chain) alleles, and drs2 mutant phenotypes such as the accumulation of enlarged Golgi cisternae, a deficiency of CCVs, and the mislocalization of TGN resident proteins. These phenotypes are similar to what is observed in clathrin mutants (Chen et al, 1999).These observations are consistent with a role for Drs2p in budding CCVs. However, because the relationship of Drs2p to well-known AP-1 and GGA clathrin adaptors has not been characterized, the precise role of Drs2p in the function of clathrin and its adaptors in the TGN-endosomal system is unclear.…”

supporting

confidence: 79%

“…This Drs2-dependent flippase activity also appears to contribute to the asymmetric distribution of endogenous PS and PE to the cytosolic leaflet as drs2⌬ mutants exhibit a loss of PS and PE asymmetry (Pomorski et al, 2003;Chen et al, 2006). P4-ATPases also play essential roles in protein transport in the secretory and endocytic pathways (Chen et al, 1999;Hua et al, 2002;Hua and Graham, 2003;Pomorski et al, 2003;Sakane et al, 2006;Furuta et al, 2007). For example, inactivation of Drs2-ts in vivo rapidly blocks formation of a clathrin-dependent class of post-Golgi vesicles carrying exocytic cargo (Gall et al, 2002).…”

mentioning

confidence: 99%

“…The protein G Sepharose beads were washed three times with washing buffer I (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 350 mM NaCl, 1% Tween-20, 1ϫ complete protease inhibitor cocktail lacking EDTA) and twice with washing buffer II (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1ϫ complete protease inhibitor cocktail lacking EDTA). Bound material was eluted with SDS/urea sample buffer, separated by SDS-PAGE, and subjected to immunoblotting as previously described (Chen et al, 1999). …”

mentioning

confidence: 99%

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

Liu

Surendhran

Nothwehr

et al. 2008

MBoC

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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 ...

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supporting

confidence: 79%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Liu

Surendhran

Nothwehr

et al. 2008

MBoC

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158

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“…DRS2, which was first isolated as a mutation that is synthetically lethal with a mutation in ARF1 encoding the Arf1p (ADP-ribosylation factor) small GTPase, has been implicated in the formation of clathrin-coated vesicles from the TGN [11]. Cdc50p has been shown to be involved in endocytic recycling of Snc1p, an exocytotic v-SNARE [13].…”

Section: Discussionmentioning

confidence: 99%

“…Drs2p localizes to endosomes and the trans-Golgi network (TGN), and has been implicated in the formation of transport vesicles from the TGN [9,11]. Cdc50p, a member of the conserved membrane-spanning protein family, was identified as a gene required for polarized cell growth [12].…”

Section: Introductionmentioning

confidence: 99%

Mutational analysis of the Lem3p-Dnf1p putative phospholipid-translocating P-type ATPase reveals novel regulatory roles for Lem3p and a carboxyl-terminal region of Dnf1p independent of the phospholipid-translocating activity of Dnf1p in yeast

Noji

Yamamoto²,

Saito³

et al. 2006

Biochemical and Biophysical Research Communications

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Lem3p-Dnf1p is a putative aminophospholipid translocase (APLT) complex that is localized to the plasma membrane; Lem3p is required for Dnf1p localization to the plasma membrane. We have identified lem3 mutations, which did not affect formation or localization of the Lem3p-Dnf1p complex, but caused a synthetic growth defect with the null mutation of CDC50, a structurally and functionally redundant homologue of LEM3. Interestingly, these lem3 mutants exhibited nearly normal levels of NBDlabeled phospholipid internalization across the plasma membrane, suggesting that Lem3p may have other functions in addition to regulation of the putative APLT activity of Dnf1p at the plasma membrane. Similarly, deletion of the COOH-terminal cytoplasmic region of Dnf1p affected neither the localization nor the APLT activity of Dnf1p at the plasma membrane, but caused a growth defect in the cdc50∆ background.Our results suggest that the Lem3p-Dnf1p complex may play a role distinct from its plasma membrane APLT activity when it substitutes for the Cdc50p-Drs2p complex, its redundant partner in the endosomal/trans-Golgi network compartments.3

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Functional morphology of the secretory pathway organelles in yeast

Vorísek

2000

Microsc. Res. Tech.

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The glycoprotein secretory pathway of yeast serves mainly for cell surface growth and cell division. It involves a centrifugal transport of transit macromolecules among organelles, whose membranes contain resident proteins needed for driving the transport. These resident membrane proteins return by retrograde vesicular transport. Apart from this, the pathway involves endocytosis. The model yeast Saccharomyces cerevisiae and vertebrate cells were found to contain very similar gene products regulating the molecular mechanism of glycoprotein transport, and the cellular mechanism of their secretion pathways was therefore also presumed to be identical. Biochemists have postulated that, in S. cerevisiae, the translocation of peptides through the endoplasmic reticulum membranes into the lumen of ER cisternae and the core glycosylation is followed by a vector‐mediated transport into the functional cascade of the Golgi system cisternae and between them. This is the site of maturation and sorting of glycoproteins, before the ultimate transport by other vectors involving either secretion from the cells (exocytosis across the plasmalemma into the cell wall) or transport into the lysosome‐like vacuole via a prevacuolar compartment, which serves at the same time as a primary endosome. The established cellular model of secretion deals with budding yeast; interphase yeast cells, in which the secretion is limited and which predominate in exponential cultures, have not been taken into consideration. The quality of organelle imaging in S. cerevisiae ultra‐thin sections depends on the fixation technique used and on specimen contrasting by metals. The results achieved by combinations of different techniques differ mostly in the imaging of bilayers of membrane interfaces and the transparence of the matrix phase. Fixation procedures are decisive for the results of topochemical localisations of cellular antigenic components or enzyme activities, which form the basis of the following survey of functional morphology of organelles involved in the yeast secretory pathway. The existing results of these studies do not confirm all aspects of the vertebrate model of the Golgi apparatus proposed by molecular geneticists to hold for S. cerevisiae, and alternative models of the cellular mechanism of secretion in this yeast are, therefore, also discussed. Microsc. Res. Tech. 51:530–546, 2000. © 2000 Wiley‐Liss, Inc.

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Role for Drs2p, a P-Type Atpase and Potential Aminophospholipid Translocase, in Yeast Late Golgi Function

Cited by 245 publications

References 65 publications

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

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

Mutational analysis of the Lem3p-Dnf1p putative phospholipid-translocating P-type ATPase reveals novel regulatory roles for Lem3p and a carboxyl-terminal region of Dnf1p independent of the phospholipid-translocating activity of Dnf1p in yeast

Functional morphology of the secretory pathway organelles in yeast

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