The F-Box Protein Rcy1p Is Involved in Endocytic Membrane Traffic and Recycling Out of an Early Endosome in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, endocytic material is transported through different membrane-bound compartments before it reaches the vacuole. In a screen for mutants that affect membrane trafficking along the endocytic pathway, we have identified a novel mutant disrupted for the gene YJL204c that we have renamed RCY1 (recycling 1). Deletion of RCY1 leads to an early block in the endocytic pathway before the intersection with the vacuolar protein sorting pathway. Mutation of RCY1 leads to the accumulation of an enlarged compartment that contains the t-SNARE Tlg1p and lies close to areas of cell expansion. In addition, endocytic markers such as Ste2p and the fluorescent dyes, Lucifer yellow and FM4-64, were found in a similar enlarged compartment after their internalization. To determine whether rcy1Δ is defective for recycling, we have developed an assay that measures the recycling of previously internalized FM4-64. This method enables us to follow the recycling pathway in yeast in real time. Using this assay, it could be demonstrated that recycling of membranes is rapid in S. cerevisiae and that a major fraction of internalized FM4-64 is secreted back into the medium within a few minutes. The rcy1Δ mutant is strongly defective in recycling.

Mutants defective in secretory/vacuolar pathways in the EUROFAN collection of yeast disruptants

“…Eight had mutations in genes identified since 1996 as being involved in trafficking (VPS53, VAC7, VAM6, APM3, SYS1) or glycosylation (ALG12, ALG9, ROT2), providing multiple internal controls of the validity of our approach. Two (TLG2, RCY1) were identified within the EUROFAN secretion node simultaneously by our Western immunoblotting approach, and by screening for defects in fluid-phase endocytosis, and we have reported the initial characterization of these mutants (Séron et al, 1998;Wiederkehr et al, 2000). One gene (MON2) was identified within the EUROFAN secretion node by the drug sensitivity approach (Muren et al, 2001).…”

“…Later analysis showed that this gene was involved in a recycling pathway, as shown by the exit of the vital dye FM4-64, following its internalization by endocytosis. This new recycling pathway is thought to involve sorting from endosomes (possibly early or 'sorting' endosomes) back to the trans-Golgi network (TGN) (Wiederkehr et al, 2000). Rcy1p is located in zones of active polarized membrane growth (Galan et al, 2001).…”

“…The Golgi hypoglycosylation defect observed in rcy1D cells may indeed result from a defect in the recycling of late Golgi enzymes (Mnn1p? ), transiting through a sorting/early endosome back to the late Golgi compartment as part of their normal cellular itinerary (Wiederkehr et al, 2000). A recycling defect may also be responsible for the hypoglycosylation observed in a subset of the other deletants.…”

“…Tlg2D cells have been reported to display defective CPY processing, reduced vacuolar uptake of LY, and retarded degradation of several plasma membrane proteins. Tlg2p was more recently described as being required for cytoplasm-to-vacuole trafficking (Abeliovich et al, 1999) and for a recycling pathway (Wiederkehr et al, 2000). It is not yet entirely clear whether this pathway originates from early or late endosomes (Galan et al, 2001;Lewis et al, 2000;Panek et al, 2000;Wiederkehr et al, 2000).…”

“…Tlg2p was more recently described as being required for cytoplasm-to-vacuole trafficking (Abeliovich et al, 1999) and for a recycling pathway (Wiederkehr et al, 2000). It is not yet entirely clear whether this pathway originates from early or late endosomes (Galan et al, 2001;Lewis et al, 2000;Panek et al, 2000;Wiederkehr et al, 2000). The distribution of Tlg2p has been reported to overlap with those of TGN and endosomal markers (Abeliovich et al, 1998;Conibear and Stevens, 2000;Holthuis et al, 1998;Panek et al, 2000;Séron et al, 1998).…”