A major challenge for a molecular understanding of membrane trafficking has been the elucidation of high-resolution structures of large, multisubunit tethering complexes that spatially and temporally control intracellular membrane fusion. Exocyst is a large hetero-octameric protein complex proposed to tether secretory vesicles at the plasma membrane to provide quality control of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion. Breakthroughs in methodologies, including sample preparation, biochemical characterization, fluorescence microscopy, and single-particle cryoelectron microscopy, are providing critical insights into the structure and function of the exocyst. These studies now pose more questions than answers for understanding fundamental functional mechanisms, and they open wide the door for future studies to elucidate interactions with protein and membrane partners, potential conformational changes, and molecular insights into tethering reactions.

Highlights Chitin and β-1,3-glucan synthases are transported separately in chitosomes and macrovesicles. Chitin synthases occupy the core of the SPK; β-1,3-glucan synthases the outer layer. CHS-4 arrival to the SPK and septa is CSE-7 dependent. Rabs YPT-1 and YPT-31 localization at the SPK mimics that of chitosomes and macrovesicles. The exocyst acts as a tether between the SPK outer layer vesicles and the apical PM.

Filamentous fungi have proven to be a better-suited model system than unicellular yeasts in analyses of cellular processes such as polarized growth, exocytosis, endocytosis, and cytoskeleton-based organelle traffic. For example, the filamentous fungus Neurospora crassa develops a variety of cellular forms. Studying the molecular basis of these forms has led to a better, yet incipient, understanding of polarized growth. Polarity factors as well as Rho GTPases, septins, and a localized delivery of vesicles are the central elements described so far that participate in the shift from isotropic to polarized growth. The growth of the cell wall by apical biosynthesis and remodeling of polysaccharide components is a key process in hyphal morphogenesis. The coordinated action of motor proteins and Rab GTPases mediates the vesicular journey along the hyphae toward the apex, where the exocyst mediates vesicle fusion with the plasma membrane. Cytoplasmic microtubules and actin microfilaments serve as tracks for the transport of vesicular carriers as well as organelles in the tubular cell, contributing to polarization. In addition to exocytosis, endocytosis is required to set and maintain the apical polarity of the cell. Here, we summarize some of the most recent breakthroughs in hyphal morphogenesis and apical growth in N. crassa and the emerging questions that we believe should be addressed.

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Vesicles in eukaryotic cells act as intracellular carriers to transport cargo between membrane‐bound organelles and the plasma membrane. Cellular trafficking is a highly regulated process that is critical for maintaining cellular growth, homeostasis, signaling and division. These cellular trafficking events ultimately culminate in membrane fusion, where secretory vesicles release their contents to the extracellular space. However, the molecular and structural underpinnings of trafficking and membrane fusion still remains misunderstood. The process of intracellular membrane fusion relies on several conserved protein families, including SNAREs, Sec1/Munc18 (SM) proteins, multi‐subunit tethering complexes (e.g. exocyst), and Rab GTPases. Each of these regulatory factors plays multiple roles in assisting vesicle docking and SNARE assembly, indicating a trafficking system with overlapping sets of reactions. Exocyst, a hetero‐octameric protein complex, is proposed to tether vesicles to the plasma membrane and promote specific SNARE‐mediated membrane fusion. Previous binding experiments using recombinant proteins revealed interactions between two exocytic SNARE proteins and Sec1 with a single subunit of exocyst, Sec6. Currently, Sec6 is proposed to bind both binary (Sso1:Sec9), and ternary (Sso1:Sec9:Snc2) SNARE complexes during SNARE complex assembly. The timing in which Sec1 binds to the ternary SNARE complex and exocyst to mediate membrane fusion is unknown. Temperature sensitive sec1 yeast cells are defective for cell growth, SNARE complex assembly, and secretion of protein and lipid cargo at the restrictive temperature. This evidence suggests that Sec1 may play both a general and a specific role during membrane trafficking. Recent structural evidence from a mutant “activated” yeast exocyst complex shows increased flexibility and dynamics of two subunits of exocyst, Exo70 and Sec6, which lead to increased binding to SNAREs compared to wild‐type exocyst. Our study aims to further investigate the mechanisms of SNARE assembly and membrane fusion, using biochemical and EM methods to characterize the interactions of yeast exocyst, Sec1 and SNARE proteins. Using purified forms of each of the proteins and complexes, interactions between Sec1, SNARE proteins, and exocyst (wild type and activated mutant) are being analyzed and quantified.