Eukaryotic cells have a highly evolved system of protein secretion, and dysfunction in this pathway is associated with many diseases including cancer, infection, metabolic disease and neurological disorders. Most proteins are secreted using the conventional endoplasmic reticulum (ER)/Golgi network and as such, this pathway is well-characterised. However, several cytosolic proteins have now been documented as secreted by unconventional transport pathways. This review focuses on two of these proteins families: annexins and galectins. The extracellular functions of these proteins are well documented, as are associations of their perturbed secretion with several diseases. However, the mechanisms and regulation of their secretion remain poorly characterised, and are discussed in this review. This review is part of a Special Issues of SCDB on 'unconventional protein secretion' edited by Walter Nickel and Catherine Rabouille.

Most secreted proteins are transported through the “conventional” endoplasmic reticulum–Golgi apparatus exocytic route for their delivery to the cell surface and release into the extracellular space. Nonetheless, formative discoveries have underscored the existence of alternative or “unconventional” secretory routes, which play a crucial role in exporting a diverse array of cytosolic proteins outside the cell in response to intrinsic demands, external cues, and environmental changes. In this context, lysosomes emerge as dynamic organelles positioned at the crossroads of multiple intracellular trafficking pathways, endowed with the capacity to fuse with the plasma membrane and recognized for their key role in both conventional and unconventional protein secretion. The recent recognition of lysosomal transport and exocytosis in the unconventional secretion of cargo proteins provides new and promising insights into our understanding of numerous physiological processes.

Galectins are a family of lectin binding proteins expressed both intracellularly and extracellularly. Galectin-3 (Gal-3, also known as LGALS3) is expressed at the cell surface; however, Gal-3 lacks a signal sequence, and the mechanism of Gal-3 transport to the cell surface remains poorly understood. Here, using a genome-wide CRISPR/Cas9 forward genetic screen for regulators of Gal-3 cell surface localization, we identified genes encoding glycoproteins, enzymes involved in N-linked glycosylation, regulators of ER-Golgi trafficking and proteins involved in immunity. The results of this screening approach led us to address the controversial role of N-linked glycosylation in the transport of Gal-3 to the cell surface. We find that N-linked glycoprotein maturation is not required for Gal-3 transport from the cytosol to the extracellular space, but is important for cell surface binding. Additionally, secreted Gal-3 is predominantly free and not packaged into extracellular vesicles. These data support a secretion pathway independent of N-linked glycoproteins and extracellular vesicles.

Cytotoxic lymphocytes eliminate virally infected or neoplastic cells through the action of cytotoxic proteases (granzymes). The pore-forming protein perforin is essential for delivery of granzymes into the cytoplasm of target cells; however the mechanism of this delivery is incompletely understood. Perforin contains a membrane attack complex/perforin (MACPF) domain and oligomerizes to form an aqueous pore in the plasma membrane; therefore the simplest (and best supported) model suggests that granzymes passively diffuse through the perforin pore into the cytoplasm of the target cell. Here we demonstrate that perforin preferentially delivers cationic molecules while anionic and neutral cargoes are delivered inefficiently. Furthermore, another distantly related pore-forming MACPF protein, pleurotolysin (from the oyster mushroom), also favors the delivery of cationic molecules, and efficiently delivers human granzyme B. We propose that this facilitated diffusion is due to conserved features of oligomerized MACPF proteins, which may include an anionic lumen.