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Novel Golgi to vacuole delivery pathway in yeast: identification of a sorting determinant and required transport
C R Cowles1, W B Snyder, C G Burd
1Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla 92093-0668, USA.
Abstract:
More than 40 vacuolar protein sorting (vps) mutants have been identified which secrete proenzyme forms of soluble vacuolar hydrolases to the cell surface. A subset of these mutants has been found to show selective defects in the sorting of two vacuolar membrane proteins. Under non-permissive conditions, vps45tsf (SEC1 homolog) and pep12/vps6tsf (endosomal t-SNARE) mutants efficiently sort alkaline phosphatase (ALP) to the vacuole while multiple soluble vacuolar proteins and the membrane protein carboxypeptidase yscS (CPS) are no longer delivered to the vacuole. Vacuolar localization of ALP in these mutants does not require transport to the plasma membrane followed by endocytic uptake, as double mutants of pep12tsf and vps45tsf with sec1 and end3 sort and mature ALP at the non-permissive temperature. Given the demonstrated role of t-SNAREs such as Pep12p in transport vesicle recognition, our results indicate that ALP and CPS are packaged into distinct transport intermediates. Consistent with ALP following an alternative route to the vacuole, isolation of a vps41tsf mutant revealed that at non-permissive temperature ALP is mislocalized while vacuolar delivery of CPS and CPY is maintained. A series of domain-swapping experiments was used to define the sorting signal that directs selective packaging and transport of ALP. Our data demonstrate that the amino-terminal 16 amino acid portion of the ALP cytoplasmic tail domain contains a vacuolar sorting signal which is responsible for the active recognition, packaging and transport of ALP from the Golgi to the vacuole via a novel delivery pathway.
Insights
Vacuolar protein sorting (VPS) mutants reveal distinct pathways for vacuolar hydrolases. Alkaline phosphatase (ALP) uses a novel Golgi-to-vacuole route mediated by its cytoplasmic tail, separate from other vacuolar proteins.
Area of Science:
- Cell Biology
- Molecular Biology
- Protein Sorting
Background:
- Vacuolar protein sorting (VPS) is crucial for cellular function, with over 40 identified VPS mutants affecting hydrolase secretion.
- Specific VPS mutants exhibit selective defects in sorting vacuolar membrane proteins, indicating complex pathway regulation.
- Understanding these pathways is key to deciphering cellular trafficking and protein localization.
Purpose of the Study:
- To investigate the distinct vacuolar sorting mechanisms for different proteins, specifically alkaline phosphatase (ALP) and carboxypeptidase yscS (CPS).
- To identify the sorting signals responsible for the selective transport of ALP.
- To elucidate a potential novel delivery pathway for ALP from the Golgi to the vacuole.
Main Methods:
- Analysis of temperature-sensitive vacuolar protein sorting (vps) mutants, including vps45tsf, pep12/vps6tsf, and vps41tsf.
- Double mutant analysis to assess the requirement of specific transport steps for ALP localization.
- Domain-swapping experiments to pinpoint the vacuolar sorting signal within ALP.
Main Results:
- Mutants defective in endosomal t-SNAREs (pep12/vps6tsf) and SEC1 homolog (vps45tsf) efficiently sort ALP but mislocalize CPS and other soluble vacuolar proteins.
- ALP vacuolar localization in these mutants does not depend on plasma membrane transport and endocytosis.
- The N-terminal 16 amino acids of the ALP cytoplasmic tail contain a functional vacuolar sorting signal, directing its transport via a novel pathway.
Conclusions:
- Alkaline phosphatase (ALP) and carboxypeptidase yscS (CPS) are sorted into distinct transport intermediates.
- ALP utilizes a novel, direct Golgi-to-vacuole pathway mediated by its cytoplasmic tail sorting signal.
- This study reveals a new mechanism for vacuolar protein delivery, distinct from previously characterized routes.