Oligomeric Dop1p is Part of the Endosomal Neo1p‐Ysl2p‐Arl1p Membrane Remodeling Complex

• Published in: Traffic (Copenhagen, Denmark) Vol. 11; no. 8; pp. 1092 - 1106
• Main Authors: Barbosa, Sónia, Pratte, Dagmar, Schwarz, Heinz, Pipkorn, Rüdiger, Singer‐Krüger, Birgit
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.08.2010
• Subjects: Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Arl1; Cell Membrane - chemistry; Cell Membrane - metabolism; endosomes; Endosomes - chemistry; Endosomes - metabolism; flippases; Gene Expression Regulation, Fungal; Intracellular Membranes - metabolism; membrane traffic; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Monomeric GTP-Binding Proteins - genetics; Monomeric GTP-Binding Proteins - metabolism; Multiprotein Complexes - chemistry; Multiprotein Complexes - metabolism; Mutation; Phospholipid Transfer Proteins; P‐type ATPase; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae - cytology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; TGN; trans-Golgi Network - metabolism; Vesicular Transport Proteins - genetics; Vesicular Transport Proteins - metabolism
• ISSN: 1398-9219; 1600-0854
• DOI: 10.1111/j.1600-0854.2010.01079.x

Yeast Dop1p is an essential protein that is highly conserved in evolution and whose function is largely unknown. Here, we provide evidence that Dop1p localizes to endosomes and exists in a complex with two other conserved proteins: Neo1p, a P4‐ATPase and putative flippase, and the scaffolding protein Ysl2p/Mon2p. The latter operates during membrane budding at the tubular endosomal network/trans‐Golgi network (TEN/TGN) in a process that includes clathrin recruitment via adaptor proteins. Consistent with a role for Dop1p during this process, temperature‐sensitive dop1‐3 cells accumulate multivesicular, elongated tubular and ring‐like structures similar to those displayed by neo1 and ysl2 mutants. In further agreement with the concept of Dop1p‐Neo1p‐Ysl2p complex formation and co‐operation, we show that dop1‐3 cells exhibit reduced levels of Neo1p and Ysl2p at steady state. Conversely, mutations or deletions in NEO1 and YSL2 lead to a decrease in Dop1p levels. In addition to binding to Neo1p and Ysl2p, Dop1p can form dimers or multimers. A critical region for dimerization resides in the C‐terminus with leucine zipper‐like domains. Dop1p's membrane association is largely mediated by its internal region, but Ysl2p might not be crucial for membrane recruitment.