Abstract
Trs20p is a subunit of the evolutionarily conserved TRAPP (TRAnsport Protein Particle) complex that mediates various aspects of membrane trafficking. Three TRAPP complexes have been identified in yeast with roles in ER-to-Golgi trafficking, post-Golgi and endosomal-to-Golgi transport and in autophagy. The role of Trs20p, which is essential for viability and a component of all three complexes, and how it might function within each TRAPP complex, has not been clarified to date. To begin to address the role of Trs20p we generated different mutants by random mutagenesis but, surprisingly, no defects were observed in diverse anterograde transport pathways or general secretion in Trs20 temperature-sensitive mutants. Instead, mutation of Trs20 led to defects in endocytic recycling and a block in sporulation/meiosis. The phenotypes of different mutants appear to be separable suggesting that the mutations affect the function of Trs20 in different TRAPP complexes.
Highlights
The transport of proteins and lipids via small vesicles or larger tubulovesicular structures from the endoplasmic reticulum (ER) to the Golgi and thence to intracellular compartments or the plasma membrane (PM) is a complex and highly regulated process
Numerous lines of evidence show that these tagged mutants represent bone fide conditional mutants of Trs20: (i) the GFP-tagged WT protein does not show any of the phenotypes associated with the mutant proteins, (ii) the tagged mutant proteins rescue the lethality caused by deletion of the Trs20 gene, (iii) coexpression of the WT Trs20 gene rescues all of the mutant phenotypes, (iv) the mutants show the same genetic interactions described in high-throughput genetic interaction screens, and (v) the untagged mutant versions show a subset of the phenotypes of the tagged mutants
The constitutive mislocalization of Snc1p and calcofluor white (CFW) hypersensitivity observed in the untagged mutants suggest that these represent functions distinct from those affected by the ts phenotype
Summary
The transport of proteins and lipids via small vesicles or larger tubulovesicular structures from the endoplasmic reticulum (ER) to the Golgi and thence to intracellular compartments or the plasma membrane (PM) is a complex and highly regulated process. SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) present in the donor and acceptor membranes mediate membrane fusion and show compartment-specific localization that confers specificity on the fusion reaction [1]. Other factors, called tethering factors, are important in conferring specificity in membrane-membrane pairing at a stage before the SNARE-mediated fusion reaction. Additional TRAPP subunits have been identified: a Trs20-like protein (Tca17/TRAPPC2L) present in both yeast and mammals that appears to be part of TRAPP II [6], [7] and mammalian-specific subunits C4orf and TTC-15 that operate in ER-to-Golgi trafficking [8]
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