Thomas Simmen

Increased ER–mitochondrial coupling promotes mitochondrial respiration and bioenergetics during early phases of ER stress

Increasing evidence indicates that endoplasmic reticulum (ER) stress activates the adaptive unfolded protein response (UPR), but that beyond a certain degree of ER damage, this response triggers apoptotic pathways. The general mechanisms of the UPR and its apoptotic pathways are well characterized. However, the metabolic events that occur during the adaptive phase of ER stress, before the cell death response, remain unknown. Here, we show that, during the onset of ER stress, the reticular and mitochondrial networks are redistributed towards the perinuclear area and their points of connection are increased in a microtubule-dependent fashion. A localized increase in mitochondrial transmembrane potential is observed only in redistributed mitochondria, whereas mitochondria that remain in other subcellular zones display no significant changes. Spatial re-organization of these organelles correlates with an increase in ATP levels, oxygen consumption, reductive power and increased mitochondrial Ca2+ uptake. Accordingly, uncoupling of the organelles or blocking Ca2+ transfer impaired the metabolic response, rendering cells more vulnerable to ER stress. Overall, these data indicate that ER stress induces an early increase in mitochondrial metabolism that depends crucially upon organelle coupling and Ca2+ transfer, which, by enhancing cellular bioenergetics, establishes the metabolic basis for the adaptation to this response.

Manipulation of oxidative protein folding and PDI redox state in mammalian cells

In the endoplasmic reticulum (ER), disulfide bonds are simultaneously formed in nascent proteins and removed from incorrectly folded or assembled molecules. In this compartment, the redox state must be, therefore, precisely regulated. Here we show that both human Ero1‐Lα and Ero1‐Lβ (hEROs) facilitate disulfide bond formation in immunoglobulin subunits by selectively oxidizing PDI. Disulfide bond formation is controlled by hEROs, which stand at a crucial point of an electron‐flow starting from nascent secretory proteins and passing through PDI. The redox state of ERp57, another ER‐resident oxidoreductase, is not affected by over‐expression of Ero1‐Lα, suggesting that parallel and specific pathways control oxidative protein folding in the ER. Mutants in the Ero1‐Lα CXXCXXC motif act as dominant negatives by limiting immunoglobulin oxidation. PDI‐dependent oxidative folding in living cells can thus be manipulated by using hERO variants.

ERp44, a novel endoplasmic reticulum folding assistant of the thioredoxin family

In human cells, Ero1‐Lα and ‐Lβ (hEROs) regulate oxidative protein folding by selectively oxidizing protein disulfide isomerase. Specific protein–protein interactions are probably crucial for regulating the formation, isomerization and reduction of disulfide bonds in the endoplasmic reticulum (ER). To identify molecules involved in ER redox control, we searched for proteins interacting with Ero1‐Lα. Here, we characterize a novel ER resident protein (ERp44), which contains a thioredoxin domain with a CRFS motif and is induced during ER stress. ERp44 forms mixed disulfides with both hEROs and cargo folding intermediates. Whilst the interaction with transport‐competent Ig‐K chains is transient, ERp44 binds more stably with J chains, which are retained in the ER and eventually degraded by proteasomes. ERp44 does not bind a short‐lived ribophorin mutant lacking cysteines. Its overexpression alters the equilibrium of the different Ero1‐Lα redox isoforms, suggesting that ERp44 may be involved in the control of oxidative protein folding.

Thiol-mediated protein retention in the endoplasmic reticulum: the role of ERp44

Formation of disulfide bonds, an essential step for the maturation and exit of secretory proteins from the endoplasmic reticulum (ER), is controlled by specific ER‐resident enzymes. A pivotal element in this process is Ero1α, an oxidoreductin that lacks known ER retention motifs. Here we show that ERp44 mediates Ero1α ER localization through the formation of reversible mixed disulfides. ERp44 also prevents the secretion of an unassembled cargo protein with unpaired cysteines. We conclude that ERp44 is a key element in thiol‐mediated retention. It might also favour the maturation of disulfide‐linked oligomeric proteins and their quality control.

Oxidative protein folding in the endoplasmic reticulum: tight links to the mitochondria-associated membrane (MAM).

The production of secretory proteins at the ER (endoplasmic reticulum) depends on a ready supply of energy and metabolites as well as the close monitoring of the chemical conditions that favor oxidative protein folding. ER oxidoreductases and chaperones fold nascent proteins into their export-competent three-dimensional structure. Interference with these protein folding enzymes leads to the accumulation of unfolded proteins within the ER lumen, causing an acute organellar stress that triggers the UPR (unfolded protein response). The UPR increases the transcription of ER chaperones commensurate with the load of newly synthesized proteins and can protect the cell from ER stress. Persistant stress, however, can force the UPR to commit cells to undergo apoptotic cell death, which requires the emptying of ER calcium stores. Conversely, a continuous ebb and flow of calcium occurs between the ER and mitochondria during resting conditions on a domain of the ER that forms close contacts with mitochondria, the MAM (mitochondria-associated membrane). On the MAM, ER folding chaperones such as calnexin and calreticulin and oxidoreductases such as ERp44, ERp57 and Ero1alpha regulate calcium flux from the ER through reversible, calcium and redox-dependent interactions with IP3Rs (inositol 1,4,5-trisphophate receptors) and with SERCAs (sarcoplasmic/endoplasmic reticulum calcium ATPases). During apoptosis progression and depending on the identity of the ER chaperone and oxidoreductase, these interactions increase or decrease, suggesting that the extent of MAM targeting of ER chaperones and oxidoreductases could shift the readout of ER-mitochondria calcium exchange from housekeeping to apoptotic. However, little is known about the cytosolic factors that mediate the on/off interactions between ER chaperones and oxidoreductases with ER calcium channels and pumps. One candidate regulator is the multi-functional molecule PACS-2 (phosphofurin acidic cluster sorting protein-2). Recent studies suggest that PACS-2 mediates localization of a mobile pool of calnexin to the MAM in addition to regulating homeostatic ER calcium signaling as well as MAM integrity. Together, these findings suggest that cytosolic, membrane and lumenal proteins combine to form a two-way switch that determines the rate of protein secretion by providing ions and metabolites and that appears to participate in the pro-apoptotic ER-mitochondria calcium transfer.

The Subcellular Distribution of Calnexin Is Mediated by PACS-2

Calnexin is an endoplasmic reticulum (ER) lectin that mediates protein folding on the rough ER. Calnexin also interacts with ER calcium pumps that localize to the mitochondria-associated membrane (MAM). Depending on ER homeostasis, varying amounts of calnexin target to the plasma membrane. However, no regulated sorting mechanism is so far known for calnexin. Our results now describe how the interaction of calnexin with the cytosolic sorting protein PACS-2 distributes calnexin between the rough ER, the MAM, and the plasma membrane. Under control conditions, more than 80% of calnexin localizes to the ER, with the majority on the MAM. PACS-2 knockdown disrupts the calnexin distribution within the ER and increases its levels on the cell surface. Phosphorylation by protein kinase CK2 of two calnexin cytosolic serines (Ser554/564) reduces calnexin binding to PACS-2. Consistent with this, a Ser554/564 Asp phosphomimic mutation partially reproduces PACS-2 knockdown by increasing the calnexin signal on the cell surface and reducing it on the MAM. PACS-2 knockdown does not reduce retention of other ER markers. Therefore, our results suggest that the phosphorylation state of the calnexin cytosolic domain and its interaction with PACS-2 sort this chaperone between domains of the ER and the plasma membrane.

Palmitoylated TMX and calnexin target to the mitochondria-associated membrane

The mitochondria‐associated membrane (MAM) is a domain of the endoplasmic reticulum (ER) that mediates the exchange of ions, lipids and metabolites between the ER and mitochondria. ER chaperones and oxidoreductases are critical components of the MAM. However, the localization motifs and mechanisms for most MAM proteins have remained elusive. Using two highly related ER oxidoreductases as a model system, we now show that palmitoylation enriches ER‐localized proteins on the MAM. We demonstrate that palmitoylation of cysteine residue(s) adjacent to the membrane‐spanning domain promotes MAM enrichment of the transmembrane thioredoxin family protein TMX. In addition to TMX, our results also show that calnexin shuttles between the rough ER and the MAM depending on its palmitoylation status. Mutation of the TMX and calnexin palmitoylation sites and chemical interference with palmitoylation disrupt their MAM enrichment. Since ER‐localized heme oxygenase‐1, but not cytosolic GRP75 require palmitoylation to reside on the MAM, our findings identify palmitoylation as key for MAM enrichment of ER membrane proteins.

RAB32 modulates apoptosis onset and mitochondria-associated membrane (MAM) properties

The mitochondria-associated membrane (MAM) has emerged as an endoplasmic reticulum (ER) signaling hub that accommodates ER chaperones, including the lectin calnexin. At the MAM, these chaperones control ER homeostasis but also play a role in the onset of ER stress-mediated apoptosis, likely through the modulation of ER calcium signaling. These opposing roles of MAM-localized chaperones suggest the existence of mechanisms that regulate the composition and the properties of ER membrane domains. Our results now show that the GTPase Rab32 localizes to the ER and mitochondria, and we identify this protein as a regulator of MAM properties. Consistent with such a role, Rab32 modulates ER calcium handling and disrupts the specific enrichment of calnexin on the MAM, while not affecting the ER distribution of protein-disulfide isomerase and mitofusin-2. Furthermore, Rab32 determines the targeting of PKA to mitochondrial and ER membranes and through its overexpression or inactivation increases the phosphorylation of Bad and of Drp1. Through a combination of its functions as a PKA-anchoring protein and a regulator of MAM properties, the activity and expression level of Rab32 determine the speed of apoptosis onset.

Ero1α requires oxidizing and normoxic conditions to localize to the mitochondria-associated membrane (MAM)

Protein secretion from the endoplasmic reticulum (ER) requires the enzymatic activity of chaperones and oxidoreductases that fold polypeptides and form disulfide bonds within newly synthesized proteins. The best-characterized ER redox relay depends on the transfer of oxidizing equivalents from molecular oxygen through ER oxidoreductin 1 (Ero1) and protein disulfide isomerase to nascent polypeptides. The formation of disulfide bonds is, however, not the sole function of ER oxidoreductases, which are also important regulators of ER calcium homeostasis. Given the role of human Ero1α in the regulation of the calcium release by inositol 1,4,5-trisphosphate receptors during the onset of apoptosis, we hypothesized that Ero1α may have a redox-sensitive localization to specific domains of the ER. Our results show that within the ER, Ero1α is almost exclusively found on the mitochondria-associated membrane (MAM). The localization of Ero1α on the MAM is dependent on oxidizing conditions within the ER. Chemical reduction of the ER environment, but not ER stress in general leads to release of Ero1α from the MAM. In addition, the correct localization of Ero1α to the MAM also requires normoxic conditions, but not ongoing oxidative phosphorylation.

Urban planning of the endoplasmic reticulum (ER): how diverse mechanisms segregate the many functions of the ER.

Abstract The endoplasmic reticulum (ER) is the biggest organelle in most cell types, but its characterization as an organelle with a continuous membrane belies the fact that the ER is actually an assembly of several, distinct membrane domains that execute diverse functions. Almost 20years ago, an essay by Sitia and Meldolesi first listed what was known at the time about domain formation within the ER. In the time that has passed since, additional ER domains have been discovered and characterized. These include the mitochondria-associated membrane (MAM), the ER quality control compartment (ERQC), where ER-associated degradation (ERAD) occurs, and the plasma membrane-associated membrane (PAM). Insight has been gained into the separation of nuclear envelope proteins from the remainder of the ER. Research has also shown that the biogenesis of peroxisomes and lipid droplets occurs on specialized membranes of the ER. Several studies have shown the existence of specific marker proteins found on all these domains and how they are targeted there. Moreover, a first set of cytosolic ER-associated sorting proteins, including phosphofurin acidic cluster sorting protein 2 (PACS-2) and Rab32 have been identified. Intra-ER targeting mechanisms appear to be superimposed onto ER retention mechanisms and rely on transmembrane and cytosolic sequences. The crucial roles of ER domain formation for cell physiology are highlighted with the specific targeting of the tumor metastasis regulator gp78 to ERAD-mediating membranes or of the promyelocytic leukemia protein to the MAM.