Specific Retrieval of the Exocytic SNARE Snc1p from Early Yeast Endosomes

TLDR

In yeast, most endocytosed proteins are routed to the vacuole, yet a subset is recycled back to the plasma membrane. The study examines the recycling pathway of GFP‑tagged exocytic SNARE Snc1p. GFP‑Snc1p is continuously recycled via the Golgi, with internalization driven by a conserved cytoplasmic signal and transmembrane domain sequences; its recycling requires Tlg1p/Tlg2p syntaxins but not late endosome–Golgi proteins, and altered recycling signals cause accumulation in early endosome‑like puncta that contain Tlg1p, Tlg2p, and Kex2p but lack Pep12p and Sec7p, indicating direct transport from these early endosomes to the Golgi.

Abstract

Many endocytosed proteins in yeast travel to the vacuole, but some are recycled to the plasma membrane. We have investigated the recycling of chimeras containing green fluorescent protein (GFP) and the exocytic SNARE Snc1p. GFP-Snc1p moves from the cell surface to internal structures when Golgi function or exocytosis is blocked, suggesting continuous recycling via the Golgi. Internalization is mediated by a conserved cytoplasmic signal, whereas diversion from the vacuolar pathway requires sequences within and adjacent to the transmembrane domain. Delivery from the Golgi to the surface is also influenced by the transmembrane domain, but the requirements are much less specific. Recycling requires the syntaxins Tlg1p and Tlg2p but not Pep12p or proteins such as Vps4p and Vps5p that have been implicated in late endosome–Golgi traffic. Subtle changes to the recycling signal cause GFP-Snc1p to accumulate preferentially in punctate internal structures, although it continues to recycle to the surface. The internal GFP-Snc1p colocalizes with Tlg1p, and immunofluorescence and immunoelectron microscopy reveal structures that contain Tlg1p, Tlg2p, and Kex2p but lack Pep12p and Sec7p. We propose that these represent early endosomes in which sorting of Snc1p and late Golgi proteins occurs, and that transport can occur directly from them to the Golgi apparatus.