Abstract
Transport of proteins through the ALP (alkaline phosphatase) pathway to the vacuole requires the function of the AP-3 adaptor complex and Vps41p. However, unlike other adaptor protein-dependent pathways, the ALP pathway has not been shown to require additional accessory proteins or coat proteins, such as membrane recruitment factors or clathrin. Two independent genetic approaches have been used to identify new mutants that affect transport through the ALP pathway. These screens yielded new mutants in both VPS41 and the four AP-3 subunit genes. Two new VPS41 alleles exhibited phenotypes distinct from null mutants of VPS41, which are defective in vacuolar morphology and protein transport through both the ALP and CPY sorting pathways. The new alleles displayed severe ALP sorting defects, normal vacuolar morphology, and defects in ALP vesicle formation at the Golgi complex. Sequencing analysis of these VPS41 alleles revealed mutations encoding amino acid changes in two distinct domains of Vps41p: a conserved N-terminal domain and a C-terminal clathrin heavy-chain repeat (CHCR) domain. We demonstrate that the N-terminus of Vps41p is required for binding to AP-3, whereas the C-terminal CHCR domain directs homo-oligomerization of Vps41p. These data indicate that a homo-oligomeric form of Vps41p is required for the formation of ALP containing vesicles at the Golgi complex via interactions with AP-3.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adaptor Proteins, Vesicular Transport
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Alkaline Phosphatase / metabolism*
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Alleles
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Amino Acid Sequence
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Binding Sites
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Carrier Proteins / pharmacology*
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Clathrin / genetics
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Clathrin / pharmacology
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Clathrin Heavy Chains
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Dimerization
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Fungal Proteins / genetics
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Fungal Proteins / metabolism
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Fungal Proteins / pharmacology
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Membrane Proteins / genetics
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Membrane Proteins / metabolism*
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Membrane Proteins / pharmacology
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Molecular Sequence Data
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Monomeric Clathrin Assembly Proteins*
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Mutation
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Nuclear Proteins*
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Phenotype
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Protein Binding
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Protein Structure, Tertiary
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Protein Transport / drug effects
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Protein Transport / genetics
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / metabolism
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RNA-Binding Proteins / pharmacology*
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Saccharomyces cerevisiae / chemistry
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Saccharomyces cerevisiae Proteins*
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Sequence Alignment
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Transport Vesicles / drug effects
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Vesicular Transport Proteins*
Substances
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Adaptor Proteins, Vesicular Transport
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Carrier Proteins
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Clathrin
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Fungal Proteins
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Membrane Proteins
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Monomeric Clathrin Assembly Proteins
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NPL3 protein, S cerevisiae
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Nuclear Proteins
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RNA-Binding Proteins
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Saccharomyces cerevisiae Proteins
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VPS41 protein, S cerevisiae
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Vesicular Transport Proteins
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clathrin assembly protein AP180
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Clathrin Heavy Chains
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Alkaline Phosphatase