Mapping Functional Interactions in a Heterodimeric Phospholipid Pump
| Autor: | Alina Tsareva, Patrick Williamson, Joost C. M. Holthuis, Hanka Hennrich, Catheleyne F. Puts, Radhakrishnan Panatala |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
Saccharomyces cerevisiae Proteins
Protein subunit Endocytic cycle Biological Transport Active Saccharomyces cerevisiae Plasma protein binding Biology Peptide Mapping Biochemistry Protein structure Membrane Biology Phospholipid Transfer Proteins Molecular Biology Adenosine Triphosphatases Cell Biology Phospholipid transport Flippase Protein Structure Tertiary Cell biology Ectodomain Multiprotein Complexes Mutation lipids (amino acids peptides and proteins) Protein Multimerization Protein Binding Cysteine |
| Zdroj: | Journal of Biological Chemistry. 287:30529-30540 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m112.371088 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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