Functional specialization within a vesicle tethering complex: bypass of a subset of exocyst deletion mutants by Sec1p or Sec4p

The exocyst is an octameric protein complex required to tether secretory vesicles to exocytic sites and to retain ER tubules at the apical tip of budded cells. Unlike the other five exocyst genes, SEC3, SEC5, and EXO70 are not essential for growth or secretion when either the upstream activator rab, Sec4p, or the downstream SNARE-binding component, Sec1p, are overproduced. Analysis of the suppressed sec3Δ, sec5Δ, and exo70Δ strains demonstrates that the corresponding proteins confer differential effects on vesicle targeting and ER inheritance. Sec3p and Sec5p are more critical than Exo70p for ER inheritance. Although nonessential under these conditions, Sec3p, Sec5p, and Exo70p are still important for tethering, as in their absence the exocyst is only partially assembled. Sec1p overproduction results in increased SNARE complex levels, indicating a role in assembly or stabilization of SNARE complexes. Furthermore, a fraction of Sec1p can be coprecipitated with the exoycst. Our results suggest that Sec1p couples exocyst-mediated vesicle tethering with SNARE-mediated docking and fusion

ER-Phagy, ER Homeostasis, and ER Quality Control:Implications for Disease

The lysosomal degradation of endoplasmic reticulum (ER) fragments by autophagy, called ER-phagy or reticulophagy, occurs under normal as well as stress conditions. The recent discovery of multiple ER-phagy receptors has stimulated studies on the roles of ER-phagy. Here, we discuss how the ER-phagy receptors and the cellular components that work with these receptors mediate two important functions: ER homeostasis and ER quality control. We highlight that ER-phagy plays an important role in alleviating ER expansion induced by ER stress and acts as an alternate disposal pathway for misfolded proteins. We suggest that the latter function explains the emerging connection between ER-phagy and disease. Additional ER-phagy-associated functions and important unanswered questions are also discussed

ER-phagy requires the assembly of actin at sites of contact between the cortical ER and endocytic pits

Fragments of the endoplasmic reticulum (ER) are selectively delivered to the lysosome (mammals) or vacuole (yeast) in response to starvation or the accumulation of misfolded proteins through an autophagic process known as ER-phagy. A screen of the Saccharomyces cerevisiae deletion library identified end3Δ as a candidate knockout strain that is defective in ER-phagy during starvation conditions, but not bulk autophagy. We find that loss of End3 and its stable binding partner Pan1, or inhibition of the Arp2/3 complex that is coupled by the End3-Pan1 complex to endocytic pits, blocks the association of the cortical ER autophagy receptor, Atg40, with the autophagosomal assembly scaffold protein Atg11. The membrane contact site module linking the rim of cortical ER sheets and endocytic pits, consisting of Scs2 or Scs22, Osh2 or Osh3, and Myo3 or Myo5, is also needed for ER-phagy. Both Atg40 and Scs2 are concentrated at the edges of ER sheets and can be cross-linked to each other. Our results are consistent with a model in which actin assembly at sites of contact between the cortical ER and endocytic pits contributes to ER sequestration into autophagosomes.</p

Mutants in trs120 disrupt traffic from the early endosome to the late Golgi

Transport protein particle (TRAPP), a large complex that mediates membrane traffic, is found in two forms (TRAPPI and -II). Both complexes share seven subunits, whereas three subunits (Trs130p, -120p, and -65p) are specific to TRAPPII. Previous studies have shown that mutations in the TRAPPII-specific gene trs130 block traffic through or from the Golgi. Surprisingly, we report that mutations in trs120 do not block general secretion. Instead, trs120 mutants accumulate aberrant membrane structures that resemble Berkeley bodies and disrupt the traffic of proteins that recycle through the early endosome. Mutants defective in recycling also display a defect in the localization of coat protein I (COPI) subunits, implying that Trs120p may participate in a COPI-dependent trafficking step on the early endosomal pathway. Furthermore, we demonstrate that Trs120p largely colocalizes with the late Golgi marker Sec7p. Our findings imply that Trs120p is required for vesicle traffic from the early endosome to the late Golgi

Myo4p and She3p are required for cortical ER inheritance in Saccharomyces cerevisiae

Myo4p is a nonessential type V myosin required for the bud tip localization of ASH1 and IST2 mRNA. These mRNAs associate with Myo4p via the She2p and She3p proteins. She3p is an adaptor protein that links Myo4p to its cargo. She2p binds to ASH1 and IST2 mRNA, while She3p binds to both She2p and Myo4p. Here we show that Myo4p and She3p, but not She2p, are required for the inheritance of cortical ER in the budding yeast Saccharomyces cerevisiae. Consistent with this observation, we find that cortical ER inheritance is independent of mRNA transport. Cortical ER is a dynamic network that forms cytoplasmic tubular connections to the nuclear envelope. ER tubules failed to grow when actin polymerization was blocked with the drug latrunculin A (Lat-A). Additionally, a reduction in the number of cytoplasmic ER tubules was observed in Lat-A–treated and myo4Δ cells. Our results suggest that Myo4p and She3p facilitate the growth and orientation of ER tubules

mBet3p is required for homotypic COPII vesicle tethering in mammalian cells

TRAPPI is a large complex that mediates the tethering of COPII vesicles to the Golgi (heterotypic tethering) in the yeast Saccharomyces cerevisiae. In mammalian cells, COPII vesicles derived from the transitional endoplasmic reticulum (tER) do not tether directly to the Golgi, instead, they appear to tether to each other (homotypic tethering) to form vesicular tubular clusters (VTCs). We show that mammalian Bet3p (mBet3p), which is the most highly conserved TRAPP subunit, resides on the tER and adjacent VTCs. The inactivation of mBet3p results in the accumulation of cargo in membranes that colocalize with the COPII coat. Furthermore, using an assay that reconstitutes VTC biogenesis in vitro, we demonstrate that mBet3p is required for the tethering and fusion of COPII vesicles to each other. Consistent with the proposal that mBet3p is required for VTC biogenesis, we find that ERGIC-53 (VTC marker) and Golgi architecture are disrupted in siRNA-treated mBet3p-depleted cells. These findings imply that the TRAPPI complex is essential for VTC biogenesis

Ypt1/Rab1 regulates Hrr25/CK1δ kinase activity in ER-Golgi traffic and macroautophagy.

ER-derived COPII-coated vesicles are conventionally targeted to the Golgi. However, during cell stress these vesicles also become a membrane source for autophagosomes, distinct organelles that target cellular components for degradation. How the itinerary of COPII vesicles is coordinated on these pathways remains unknown. Phosphorylation of the COPII coat by casein kinase 1 (CK1), Hrr25, contributes to the directional delivery of ER-derived vesicles to the Golgi. CK1 family members are thought to be constitutively active kinases that are regulated through their subcellular localization. Instead, we show here that the Rab GTPase Ypt1/Rab1 binds and activates Hrr25/CK1δ to spatially regulate its kinase activity. Consistent with a role for COPII vesicles and Hrr25 in membrane traffic and autophagosome biogenesis, hrr25 mutants were defective in ER–Golgi traffic and macroautophagy. These studies are likely to serve as a paradigm for how CK1 kinases act in membrane traffic