Skp1p and the F-Box Protein Rcy1p Form a Non-SCF Complex Involved in Recycling of the SNARE Snc1p in Yeast

Abstract

Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin-ligases composed of a core complex including Skp1p, Cdc53p, Hrt1p, the E2 enzyme Cdc34p, and one of multiple F-box proteins which are thought to provide substrate specificity to the complex. Here we show that the F-box protein Rcy1p is required for recycling of the v-SNARE Snc1p in Saccharomyces cerevisiae. Rcy1p localized to areas of polarized growth, and this polarized localization required its CAAX box and an intact actin cytoskeleton. Rcy1p interacted with Skp1p in vivo in an F-box-dependent manner, and both deletion of its F box and loss of Skp1p function impaired recycling. In contrast, cells deficient in Cdc53p, Hrt1p, or Cdc34p did not exhibit recycling defects. Unlike the case for F-box proteins that are known to participate in SCF complexes, degradation of Rcy1p required neither its F box nor functional 26S proteasomes or other SCF core subunits. Importantly, Skp1p was the only major partner that copurified with Rcy1p. Our results thus suggest that a complex composed of Rcy1p and Skp1p but not other SCF components may play a direct role in recycling of internalized proteins.

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

“…rcy1D cells and not tlg2D cells). This systematic analysis enabled us to identify potentially interesting genes involved in intracellular protein trafficking that had already been characterized (Galan et al, 2001;Séron et al, 1998;Wiederkehr et al, 2000). The study of YJR044c, a new VPS gene, revealed the function of a new protein family, conserved from yeast to man (Belgareh-Touzé et al, submitted).…”

“…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).…”

“…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).…”

Sgt1p and Skp1p Modulate the Assembly and Turnover of CBF3 Complexes Required for Proper Kinetochore Function

“…The turnover of Ctf13p may also contribute to the ability to create new CBF3 complexes, a process that may be linked to maintenance of the spindle checkpoint (Kitagawa et al, 2003). Interestingly, it has been reported that Skp1p is required for the turnover of Rcy1p, a protein involved in the recycling of SNARE complexes, independent of its role in SCF (Galan et al, 2001); this result and our observations concerning the stability of CBF3 in skp1-3 cells raises the possibility that Skp1p contributes to the turnover of proteins independent of ubiquitin-mediated degradation. Whether degradation of Ctf13p itself serves as the lynchpin for CBF3 turnover, or some other process involving chaperones is involved, will require a more detailed examination of how CBF3, and perhaps SCF, complexes are turned over.…”

Mcs2 and a novel CAK subunit Pmh1 associate with Skp1 in fission yeast

“…This is interesting as Skp1 functions not only as part of SCF but also plays a role as a regulator of a variety of complexes and functions including cell separation [40], recycling of proteins from the plasma membrane [59], and the assembly of vacuolar ATPases [47,60] apparently independently of SCF. Furthermore, our data suggest a potential new regulatory mechanism of Mcs2 and Pmh1 that could selectively regulate the distribution of these CAK subunits amongst the various cellular subcomplexes such as the trimeric versus TFIIH-associated forms of Mcs6.…”