Sec2p is a guanine nucleotide exchange factor that activates Sec4p, the final Rab GTPase of the yeast secretory pathway. Sec2p is recruited to secretory vesicles by the upstream Rab Ypt32p acting in concert with phosphatidylinositol-4-phosphate (PI(4)P). Sec2p also binds to the Sec4p effector Sec15p, yet Ypt32p and Sec15p compete against each other for binding to Sec2p. We report here that the redundant casein kinases Yck1p and Yck2p phosphorylate sites within the Ypt32p/Sec15p binding region and in doing so promote binding to Sec15p and inhibit binding to Ypt32p. We show that Yck2p binds to the autoinhibitory domain of Sec2p, adjacent to the PI(4)P binding site, and that addition of PI(4)P inhibits Sec2p phosphorylation by Yck2p. Loss of Yck1p and Yck2p function leads to accumulation of an intracellular pool of the secreted glucanase Bgl2p, as well as to accumulation of Golgi-related structures in the cytoplasm. We propose that Sec2p is phosphorylated after it has been recruited to secretory vesicles and the level of PI(4)P has been reduced. This promotes Sec2p function by stimulating its interaction with Sec15p. Finally, Sec2p is dephosphorylated very late in the exocytic reaction to facilitate recycling.

The endoplasmic reticulum (ER) forms a contiguous network of tubules and sheets that is predominantly associated with the cell cortex in yeast. Upon treatment with rapamycin, the ER undergoes degradation by selective autophagy. This process, termed ER-phagy, requires Atg40, a selective autophagy receptor that localizes to the cortical ER. Here we report that ER-phagy also requires Lnp1, an ER membrane protein that normally resides at the three-way junctions of the ER network, where it serves to stabilize the network as it is continually remodeled. Rapamycin treatment increases the expression of Atg40, driving ER domains marked by Atg40 puncta to associate with Atg11, a scaffold protein needed to form autophagosomes. Although Atg40 largely localizes to the cortical ER, the autophagy machinery resides in the cell interior. The localization of Atg40 to sites of autophagosome formation is blocked in an lnp1Δ mutant or upon treatment of wild-type cells with the actin-depolymerizing drug Latrunculin A. This prevents the association of Atg40 with Atg11 and the packaging of the ER into autophagosomes. We propose that Lnp1 is needed to stabilize the actin-dependent remodeling of the ER that is essential for ER-phagy.

The endoplasmic reticulum (ER) consists of a polygonal network of sheets and tubules interconnected by three-way junctions. This network undergoes continual remodeling through competing processes: the branching and fusion of tubules forms new three-way junctions and new polygons, and junction sliding and ring closure leads to polygon loss. However, little is known about the machinery required to generate and maintain junctions. We previously reported that yeast Lnp1 localizes to ER junctions, and that loss of Lnp1 leads to a collapsed, densely reticulated ER network. In mammalian cells, only approximately half the junctions contain Lnp1. Here we use live cell imaging to show that mammalian Lnp1 (mLnp1) affects ER junction mobility and hence network dynamics. Three-way junctions with mLnp1 are less mobile than junctions without mLnp1. Newly formed junctions that acquire mLnp1 remain stable within the ER network, whereas nascent junctions that fail to acquire mLnp1 undergo rapid ring closure. These findings imply that mLnp1 plays a key role in stabilizing nascent three-way ER junctions.

Endoplasmic reticulum (ER) macroautophagy (hereafter called ER-phagy) uses autophagy receptors to selectively degrade ER domains in response to starvation or the accumulation of aggregation-prone proteins. Autophagy receptors package the ER into autophagosomes by binding to the ubiquitin-like yeast protein Atg8 (LC3 in mammals), which is needed for autophagosome formation. In budding yeast, cortical and cytoplasmic ER-phagy requires the autophagy receptor Atg40. While different ER autophagy receptors have been identified, little is known about other components of the ER-phagy machinery. In an effort to identify these components, we screened the genome-wide library of viable yeast deletion mutants for defects in the degradation of cortical ER following treatment with rapamycin, a drug that mimics starvation. Among the mutants we identified was vps13Δ. While yeast has one gene that encodes the phospholipid transporter VPS13, humans have four vacuolar protein-sorting (VPS) protein 13 isoforms. Mutations in all four human isoforms have been linked to different neurological disorders, including Parkinson's disease. Our findings have shown that Vps13 acts after Atg40 engages the autophagy machinery. Vps13 resides at contact sites between the ER and several organelles, including late endosomes. In the absence of Vps13, the cortical ER marker Rtn1 accumulated at late endosomes, and a dramatic decrease in ER packaging into autophagosomes was observed. Together, these studies suggest a role for Vps13 in the sequestration of the ER into autophagosomes at late endosomes. These observations may have important implications for understanding Parkinson's and other neurological diseases.