Localization and activity of the SNARE Ykt6 determined by its regulatory domain and palmitoylation

Abstract

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze compartment-specific membrane fusion. Whereas most SNAREs are bona fide type II membrane proteins, Ykt6 lacks a proteinaceous membrane anchor but contains a prenylation consensus motif (CAAX box) and exists in an inactive cytosolic and an active membrane-bound form. We demonstrate that both forms are farnesylated at the carboxyl-terminal cysteine of the CCAIM sequence. Farnesylation is the prerequisite for subsequent palmitoylation of the upstream cysteine, which permits stable membrane association of Ykt6. The double-lipid modification and membrane association is crucial for intra-Golgi transport in vitro and cell homeostasis͞survival in vivo. The membrane recruitment and palmitoylation is controlled by the N-terminal domain of Ykt6, which interacts with the SNARE motif, keeping it in an inactive closed conformation. Together, these results suggest that conformational changes control the lipid modification and function of Ykt6. Considering the essential and central role of Ykt6 in the secretory pathway, this spatial and functional cycle might provide a mechanism to regulate the rate of intracellular membrane flow.T he dynamic and specific trafficking of proteins and lipids along the secretory and endocytic pathways relies on precisely choreographed membrane fusion events, in turn relying on the faithful pairing of cognate soluble N-ethylmaleimidesensitive factor attachment protein receptors (SNAREs) between membranes (1, 2). In vitro membrane fusion assays employing purified SNAREs reconstituted into liposomes, as well as in vivo cell-cell fusion experiments using f lipped SNAREs [cognate-vesicle (v) and target-membrane (t) SNAREs expressed on the extracellular surface of two cell populations, respectively], provide compelling evidence that SNAREs are the driving force for fusion and, in addition, encode targeting specificity (3-7). Furthermore, as reported for intraGolgi transport, some SNAREs function also as inhibitory SNAREs to fine-tune specific fusion events (8). Thus, the intracellular distribution of cognate SNAREs outlines the fusion potential of distinct compartments and provides a road map for membrane trafficking (9, 10).The rate at which compartments containing cognate SNAREs fuse is at least in part determined by the conformational state of SNAREs. Most SNAREs contain regulatory domains in addition to their SNARE motifs, which form the four-helix bundle at the core of the SNARE complex (11). These regulatory domains control the assembly of the three-helix t-SNARE (one helix derived from a syntaxin heavy chain and two from t-SNARE light chains) and͞or the subsequent v-͞t-SNARE complex formation. Additional components can either bind or posttranslationally modify SNAREs, increasing or decreasing SNARE activity.A unique and therefore interesting SNARE is Ykt6, an essential protein that is highly conserved from yeast to man (12). Ykt6 is special in that it is lipid-anchored to membranes, lacking the usual hydroph...

Systematic Analysis of SNARE Molecules in <i>Arabidopsis</i>: Dissection of the post-Golgi Network in Plant Cells

“…SYP23 (Qa) may be unable to bind to membranes due to the lack of a Cterminal TMD caused by a frame-shift mutation (Zheng et al, 1999). AtYKT61 (R) is considered to be anchored to the membrane by a post-transcriptional modification similar to the YKT6 group in yeast and animals (Fukasawa et al, 2004;McNew et al, 1997), hence GFP conjugation might interfere with the post-translational modification site of this protein.…”

Yeast vacuole fusion: A model system for eukaryotic endomembrane dynamics

“…On membranes, Ykt6 is both prenylated and palmitoylated, whereas it is believed to be depalmitoylated upon dissociation from membranes. [110][111][112] In its cytosolic state, the N-terminal longin-domain of Ykt6 is thought to fold back onto its C-terminal coiled-coil domain, thereby enveloping its hydrophobic prenyl anchor. 113 Ykt6 may regulate its own palmitoylation and depalmitoylation 114 and thereby determine its localization.…”

“…113 Ykt6 may regulate its own palmitoylation and depalmitoylation 114 and thereby determine its localization. 110,111 The function of Ykt6 at the vacuole remains puzzling. It is implicated to function in several trafficking pathways towards the vacuole, i.e.…”

Identification of the Yeast R-SNARE Nyv1p as a Novel Longin Domain-containing Protein

“…These include subunits of the adaptin complexes (Collins et al, 2002;Heldwein et al, 2004) and SEDL/Trs20p (Jang et al, 2002), a common subunit of the transport protein particle (TRAPP)I and TRAPPII multisubunit complexes, which are required for traffic between the ER and Golgi and within the Golgi (Oka and Krieger, 2005). The function of the longin fold of Sec22b/Sec22p is unknown, but in Ykt6p the longin domain folds back and binds to the SNARE-motif of the protein (Tochio et al, 2001), and this conformation is likely to be important for chaperoning the lipid modified C terminus of the cytoplasmic form of Ykt6p in cells as well as for its localization (Fukasawa et al, 2004;Hasegawa et al, 2004).…”