Membrane trafficking: ESCRTs act here, there, and everywhere.

Endosomal sorting complex required for transport (ESCRT) proteins can promote extreme membrane deformations, including scission and sealing. New work uncovers a link between these proteins and the early secretory pathway that is functionally important for programmed autophagy during Drosophila development.

Pep4-dependent microautophagy is required for post-ER degradation of GPI-anchored proteins.

Clearance of misfolded proteins from the secretory pathway often occurs soon after their biosynthesis by endoplasmic reticulum (ER)-associated protein degradation (ERAD). However, certain types of misfolded proteins are not ERAD substrates and exit the ER. They are then scrutinized by ill-defined post-ER quality control (post-ERQC) mechanisms and are frequently routed to the vacuole/lysosome for degradation.

Post-ER degradation of misfolded GPI-anchored proteins is linked with microautophagy.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are membrane-conjugated cell-surface proteins with diverse structural, developmental, and signaling functions and clinical relevance. Typically, after biosynthesis and attachment to the preassembled GPI anchor, GPI-APs rapidly leave the endoplasmic reticulum (ER) and rely on post-ER quality control. Terminally misfolded GPI-APs end up inside the vacuole/lysosome for degradation, but their trafficking itinerary to this organelle and the processes linked to their uptake by the vacuole/lysosome remain uncharacterized.

Dual Independent Roles of the p24 Complex in Selectivity of Secretory Cargo Export from the Endoplasmic Reticulum.

The cellular mechanisms that ensure the selectivity and fidelity of secretory cargo protein transport from the endoplasmic reticulum (ER) to the Golgi are still not well understood. The p24 protein complex acts as a specific cargo receptor for GPI-anchored proteins by facilitating their ER exit through a specialized export pathway in yeast. In parallel, the p24 complex can also exit the ER using the general pathway that exports the rest of secretory proteins with their respective cargo receptors.

Lipids and their (un)known effects on ER-associated protein degradation (ERAD).

Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is a conserved cellular process that apart from protein quality control and maintenance of ER membrane identity has pivotal functions in regulating the lipid composition of the ER membrane. A general trigger for ERAD activation is the exposure of normally buried protein domains due to protein misfolding, absence of binding partners or conformational changes. Several feedback loops for ER lipid homeostasis exploit the induction of conformational changes in key enzymes of lipid biosynthesis or in ER membrane-embedded transcription factors upon shortage or abundance of specific lipids, leading to enzyme degradation or mobilization of transcription factors.

Loss of glutathione redox homeostasis impairs proteostasis by inhibiting autophagy-dependent protein degradation.

In the presence of aggregation-prone proteins, the cytosol and endoplasmic reticulum (ER) undergo a dramatic shift in their respective redox status, with the cytosol becoming more oxidized and the ER more reducing. However, whether and how changes in the cellular redox status may affect protein aggregation is unknown. Here, we show that C.

Specific COPII vesicles transport ER membranes to sites of autophagosome formation.

The endoplasmic reticulum (ER) is considered a prominent membrane source for the formation of autophagosomes. Recent results from our laboratory revealed a cellular mechanism for the contribution of the ER to autophagosomes in yeast: membranes, together with unconventional membrane fusion machinery, are delivered to sites of autophagosome formation by specific coat protein complex II (COPII) vesicles.

Limited ER quality control for GPI-anchored proteins.

Endoplasmic reticulum (ER) quality control mechanisms target terminally misfolded proteins for ER-associated degradation (ERAD). Misfolded glycophosphatidylinositol-anchored proteins (GPI-APs) are, however, generally poor ERAD substrates and are targeted mainly to the vacuole/lysosome for degradation, leading to predictions that a GPI anchor sterically obstructs ERAD. Here we analyzed the degradation of the misfolded GPI-AP Gas1* in yeast.

A SNARE and specific COPII requirements define ER-derived vesicles for the biogenesis of autophagosomes.

The endoplasmic reticulum (ER) is a major source for the generation of autophagosomes during macroautophagy. Our recent work in yeast shows that particular ER-derived vesicles are generated for the biogenesis of autophagosomes. These vesicles not only incorporate a SNARE protein that is largely ER-resident under nonstarving conditions, but also display COPII requirements for ER-exit that differ from conventional cargo-transporting vesicles.

An ER-Localized SNARE Protein Is Exported in Specific COPII Vesicles for Autophagosome Biogenesis.

The de novo formation of autophagosomes for the targeting of cytosolic material to the vacuole/lysosome is upregulated upon starvation. How autophagosomes acquire membranes remains still unclear. Here, we report that, in yeast, the endoplasmic reticulum (ER)-localized Qa/t-SNARE Ufe1 has a role in autophagy.

COPII coat composition is actively regulated by luminal cargo maturation.

Background: Export from the ER is an essential process driven by the COPII coat, which forms vesicles at ER exit sites (ERESs) to transport mature secretory proteins to the Golgi. Although the basic mechanism of COPII assembly is known, how COPII machinery is regulated to meet varying cellular secretory demands is unclear.

Results: Here, we report a specialized COPII system that is actively recruited by luminal cargo maturation.

Regulation of Endoplasmic Reticulum-Associated Protein Degradation (ERAD) by Ubiquitin.

Quality control of protein folding inside the endoplasmic reticulum (ER) includes chaperone-mediated assistance in folding and the selective targeting of terminally misfolded species to a pathway called ER-associated protein degradation, or simply ERAD. Once selected for ERAD, substrates will be transported (back) into the cytosol, a step called retrotranslocation. Although still ill defined, retrotranslocation likely involves a protein conducting channel that is in part formed by specific membrane-embedded E3 ubiquitin ligases.

Roles of ubiquitin in endoplasmic reticulum-associated protein degradation (ERAD).

In the secretory pathway, quality control for the correct folding of proteins is largely occurring in the endoplasmic reticulum (ER), at the earliest possible stage and in an environment where early folding intermediates mix with terminally misfolded species. An elaborate cellular mechanism aims at dividing the former from the latter and promotes the selective transport of misfolded species back into the cytosol, a step called retrotranslocation. During retrotranslocation proteins will become ubiquitinated on the cytosolic side of the ER membrane by dedicated machineries and will be targeted to the proteasome for degradation.

Protein O-mannosyltransferases participate in ER protein quality control.

In eukaryotic cells, proteins enter the secretory pathway at the endoplasmic reticulum (ER) as linear polypeptides and fold after translocation across or insertion into the membrane. If correct folding fails, many proteins are O-mannosylated inside the ER by an O-mannosyltransferase, the Pmt1p-Pmt2p complex. The consequences of this modification are controversial and the cellular role of the Pmt1p-Pmt2p complex in this respect is unclear.

The ER-associated degradation component Der1p and its homolog Dfm1p are contained in complexes with distinct cofactors of the ATPase Cdc48p.

Misfolded proteins in the endoplasmic reticulum (ER) are often degraded in the cytosol by a process called ER-associated protein degradation (ERAD). During ERAD in S. cerevisiae, the ATPase Cdc48p associates with Der1p, a putative component of a retro-translocation channel.

Mutations in the Sec61p channel affecting signal sequence recognition and membrane protein topology.

The orientation of most single-spanning membrane proteins obeys the "positive-inside rule", i.e. the flanking region of the transmembrane segment that is more positively charged remains in the cytosol.

Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins.

Many misfolded endoplasmic reticulum (ER) proteins are eliminated by ERAD, a process in which substrates are polyubiquitylated and moved into the cytosol for proteasomal degradation. We have identified in S. cerevisiae distinct ubiquitin-ligase complexes that define different ERAD pathways.

The plug domain of yeast Sec61p is important for efficient protein translocation, but is not essential for cell viability.

The Sec61/SecY translocon mediates translocation of proteins across the membrane and integration of membrane proteins into the lipid bilayer. The structure of the translocon revealed a plug domain blocking the pore on the lumenal side. It was proposed to be important for gating the protein conducting channel and for maintaining the permeability barrier in its unoccupied state.

Membrane-protein integration and the role of the translocation channel.

Most eukaryotic membrane proteins are integrated into the lipid bilayer during their synthesis at the endoplasmic reticulum (ER). Their integration occurs with the help of a protein-conducting channel formed by the heterotrimeric Sec61 membrane-protein complex. The crystal structure of an archaeal homolog of the complex suggests mechanisms that enable the channel to open across the membrane and to release laterally hydrophobic transmembrane segments of nascent membrane proteins into lipid.

Sec61p contributes to signal sequence orientation according to the positive-inside rule.

Protein targeting to the endoplasmic reticulum is mediated by signal or signal-anchor sequences. They also play an important role in protein topogenesis, because their orientation in the translocon determines whether their N- or C-terminal sequence is translocated. Signal orientation is primarily determined by charged residues flanking the hydrophobic core, whereby the more positive end is predominantly positioned to the cytoplasmic side of the membrane, a phenomenon known as the "positive-inside rule.

Molecular mechanism of signal sequence orientation in the endoplasmic reticulum.

We have analyzed in vivo how model signal sequences are inserted and oriented in the membrane during cotranslational integration into the endoplasmic reticulum. The results are incompatible with the current models of retention of positive flanking charges or loop insertion of the polypeptide into the translocon. Instead they indicate that these N-terminal signals initially insert head-on with a cytoplasmic C-terminus before they invert their orientation to translocate the C-terminus.