Mapping Functional Interactions in a Heterodimeric Phospholipid Pump

Autor(en): Puts, Catheleyne F.
Panatala, Radhakrishnan
Hennrich, Hanka
Tsareva, Alina
Williamson, Patrick
Holthuis, Joost C. M.
Stichwörter: AMINOPHOSPHOLIPID TRANSPORT; ASYMMETRY; Biochemistry & Molecular Biology; CDC50 PROTEINS; CRYSTAL-STRUCTURE; P-TYPE ATPASES; PLASMA-MEMBRANE; ROLES; SUBCELLULAR-LOCALIZATION; TRANSLOCATION; YEAST
Erscheinungsdatum: 2012
Herausgeber: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Enthalten in: JOURNAL OF BIOLOGICAL CHEMISTRY
Band: 287
Ausgabe: 36
Startseite: 30529
Seitenende: 30540
Zusammenfassung: 
Type 4 P-type ATPases (P-4-ATPases) catalyze phospholipid transport to generate phospholipid asymmetry across membranes of late secretory and endocytic compartments, but their kinship to cation-transporting P-type transporters raised doubts about whether P-4-ATPases alone are sufficient to mediate flippase activity. P-4-ATPases form heteromeric complexes with Cdc50 proteins. Studies of the enzymatic properties of purified P-4-ATPase.Cdc50 complexes showed that catalytic activity depends on direct and specific interactions between Cdc50 subunit and transporter, whereas in vivo interaction assays suggested that the binding affinity for each other fluctuates during the transport reaction cycle. The structural determinants that govern this dynamic association remain to be established. Using domain swapping, site-directed, and random mutagenesis approaches, we here show that residues throughout the subunit contribute to forming the heterodimer. Moreover, we find that a precise conformation of the large ectodomain of Cdc50 proteins is crucial for the specificity and functionality to transporter/subunit interactions. We also identified two highly conserved disulfide bridges in the Cdc50 ectodomain. Functional analysis of cysteine mutants that disrupt these disulfide bridges revealed an inverse relationship between subunit binding and P-4-ATPase-catalyzed phospholipid transport. Collectively, our data indicate that a dynamic association between subunit and transporter is crucial for the transport reaction cycle of the heterodimer.
ISSN: 00219258
DOI: 10.1074/jbc.M112.371088

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