The polybasic juxtamembrane region of Sso1p is required for SNARE function in vivo.

Biological membrane fusion is imperative for cellular survival. Membrane fusion requires an energy-dependent reaction to overcome the repulsive nature of two opposing membranes (4, 10, 11). In cells, conformational rearrangements of specific fusion proteins drive this membrane merger (9, 44). Membrane fusion reactions in the secretory pathway have been extensively studied, and much is known about the molecular machinery, and yet many aspects of this process are not well understood. SNARE proteins (Soluble NSF attachment protein receptors) constitute the core fusion machinery (38, 49) and are the final arbiters of fusion specificity (23). SNAREs are operationally divided into two groups: those that are found primarily on the transport vesicle, called v-SNAREs, and those found primarily on the target membrane, called t-SNAREs. The founding members of the SNARE superfamily were identified from bovine brain and participate in synaptic transmission (38). The neuronal SNARE complex is comprised of two t-SNAREs localized to the plasma membrane, called syntaxin 1A (3) and SNAP25 (synaptosome-associated protein of 25 kDa [28]), as well as one v-SNARE located on the synaptic vesicle, known as VAMP (vesicle-associated membrane protein or synaptobrevin [40, 43]). SNARE proteins also share structural and mechanistic commonalities with viral fusogens, such as the assembly of a coiled-coil bundle structure and a hemifusion transition-state intermediate (15, 22, 36, 39, 51). However, several elements that regulate membrane fusion, including proteins such as Rab GTPases and the exocyst complex, remain incompletely understood (27, 30, 31, 52). Similarly, the Sec1/Munc18 family of SNARE regulators has a variety of proposed functions, including direct stimulation of fusion through interactions with the yeast plasma membrane t-SNARE complex (18, 42). The fusion of Golgi-derived transport vesicles with the plasma membrane requires the formation of a binary t-SNARE complex on the plasma membrane comprised of a syntaxin family member and the respective SNAP25 homolog. Complete membrane fusion occurs when a VAMP-like v-SNARE found on the transport vesicle associates with the t-SNARE complex to form a ternary SNARE complex. This association is thought to occur in a “zipper”-like fashion that pulls the membranes close together (7, 16, 17). The structure of the cytosolic portion of the four-stranded neuronal core complex shows that the proteins align in a parallel fashion with the transmembrane domains of both syntaxin 1A and VAMP emerging from the same end of the helical bundle (32, 41). Membrane fusion of Golgi-derived transport vesicles with the plasma membrane in yeast utilizes homologous SNARE proteins. The plasma membrane t-SNARE complex is composed of the syntaxin 1A homolog Sso1p (1) or the functionally redundant Sso2p and the SNAP25-like Sec9p (6). The v-SNARE Snc1p (14) or the functionally redundant Snc2p completes the four-helix bundle. The majority of SNARE proteins have a membrane-spanning region at their extreme carboxy terminus with a nearby core complex domain that has the propensity to form coiled coils. A common feature conserved in many if not all plasma membrane syntaxins is a polybasic juxtamembrane region between the core complex domain and the transmembrane domain (Fig. ​(Fig.1B)1B) (50). Similar stretches of charge are also seen in plasma membrane v-SNAREs. Electron paramagnetic resonance measurements suggest that the juxtamembrane regions of the neuronal SNAREs syntaxin 1A and VAMP interact with and are embedded in the phospholipid bilayer (20, 21). FIG. 1. Domain structure and sequence of Sso1p, Sso1p juxtamembrane mutations, and other plasma membrane syntaxins. A. The general domain structure of Sso1p is depicted. HA, HB, and HC refer to predicted coiled-coil domains that form an autonomously folding N-terminal ... Previous in vitro studies have shown that additional sequence in the juxtamembrane of the neuronal t-SNARE syntaxin 1A and the v-SNARE VAMP is permitted, although the amount of fusion progressively decreases as the distance between the core complex domain and the transmembrane region increases (24). The introduction of sequential helix-breaking proline residues in the juxtamembrane region of syntaxin 1A reduced fusion ∼50% in a liposome mixing assay; however, the same insertion in VAMP2 did not significantly impair fusion in vitro (24). While this study examined the in vitro function of juxtamembrane linker mutants, no in vivo analysis was possible. Saccharomyces cerevisiae is amenable to both in vitro and in vivo studies, allowing mechanistic models developed in vitro to be directly tested in vivo. The present work correlates in vivo mutational analysis of Sso1p function with in vitro fusion activity to examine the function of the juxtamembrane region of Sso1p. Modifications introducing distance and flexibility were used to test bilayer-coupling models of fusion in a reconstituted fusion assay, similar to prior work performed with neuronal SNAREs, and to examine the effects of the same mutations on in vivo SNARE function. Additional mutations examined the specific sequence requirements of the juxtamembrane region necessary to maintain in vivo function.