Edited by: Cecilia Poderoso, University of Buenos Aires, Argentina
Reviewed by: Amandine Grimm, University of Basel, Switzerland; Tito Cali, University of Padova, Italy; Charles Affourtit, University of Plymouth, United Kingdom
This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology
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Secretion is an energy consuming process that plays a relevant role in cell communication and adaptation to the environment. Among others, endocrine cells producing hormones, immune cells producing cytokines or antibodies, neurons releasing neurotransmitters at synapsis, and more recently acknowledged, senescent cells synthesizing and secreting multiple cytokines, growth factors and proteases, require energy to successfully accomplish the different stages of the secretion process. Calcium ions (Ca2+) act as second messengers regulating secretion in many of these cases. In this setting, mitochondria appear as key players providing ATP by oxidative phosphorylation, buffering Ca2+ concentrations and acting as structural platforms. These tasks also require the concerted actions of the mitochondrial dynamics machinery. These proteins mediate mitochondrial fusion and fission, and are also required for transport and tethering of mitochondria to cellular organelles where the different steps of the secretion process take place. Herein we present a brief overview of mitochondrial energy metabolism, mitochondrial dynamics, and the different steps of the secretion processes, along with evidence of the interaction between these pathways. We also analyze the role of mitochondria in secretion by different cell types in physiological and pathological settings.
About 20% of the proteins synthesized by eukaryotic cells are secreted to the extracellular space either as soluble or membrane bound proteins (
As many other complex cellular processes secretion of proteins consumes energy, therefore requires the support of functional mitochondria. In the conventional pathway proteins are transported into the endoplasmic reticulum (ER), and folding and quality control of proteins in the ER (
Work by others and us shows that impairment of mitochondrial catabolism and dynamics affects the secretion processes (
While extensive literature can be found regarding both secretion and mitochondrial bioenergetics and dynamics, the connection between these processes and the organelles involved are still largely unexplored. In this manuscript secretion pathways, mitochondrial metabolism and dynamics
Main roles for mitochondria in secretory processes. (1) Mitochondria provide ATP, obtained by oxidative phosphorylation, for: protein synthesis, translocation to the ER, folding and quality control, vesicle transport, vesicle fusion and exocytosis, Ca2+ pumping across plasma and ER membranes; and inflammasome activation. (2) Mitochondria can uptake Ca2+, modulating Ca2+ concentration and therefore vesicle exocytosis. (3) Mitochondria provide a structural scaffold for the assembly of the NLRP3 inflammasome.
Secreted proteins can reach the extracellular media through the conventional (classic) pathway or through unconventional pathways (
Mitochondria and the conventional secretion machinery. To support the energy requirements of the secretion process mitochondria interact with organelles and components of the cytoskeleton and supply ATP for: (1) Protein synthesis, translocation to the ER and folding. (2) Protein quality control in particular for the energy consuming ERAD. (3) Vesicle fusion with target membranes in the ER, ERGIC, Golgi, and plasma membrane. (4) Vesicle and mitochondrial transport along microtubules and actin filaments. (5) Exocytosis. In the panel below the figure are a series of molecules and complexes that play relevant roles in these events.
Most of the secreted proteins in the conventional pathway are translocated to the rough ER during translation by the ribosome (
The conventional pathway for protein secretion and its energy requirements.
Protein synthesis | ( |
|
Protein translocation | SRP, SRP receptor, SEC61, SEC62, SEC63, |
( |
Protein folding | OGT, exoglucosidases I and II, calnexin, calreticulin, GT, PDIA3, |
( |
Protein quality control | ER α1,2-mannosidase, EDEMs 1/2/3, |
( |
COPII and COPIvesicle assembly | SAR1, ARF, SEC12, SEC16, SEC23, SEC24, SEC13, SEC31, COPI subunits, GEFs, GAPs | ( |
Vesicle fusion | Receptors, Rab GTPases, Rab effectors SNARE proteins, SM proteins, SNAP, |
( |
Vesicle transport | ( |
|
Exocytosis | Rab GTPases, Rab effectors SNARE proteins, SM proteins, SNAP, |
( |
In the ER, the oligosaccharyl transferase complex (OGT) catalyzes the transfer of the oligosaccharide (Glc3Man9GlcNAc2) from dolichol phosphate to an asparagine residue in the protein (
In the ER proteins undergo quality control pathways for the detection, remotion, and degradation of misfolded proteins that did not attain their native structures. Degradation by the proteasome takes place in the cytosol in a pathway known as ER-associated degradation (ERAD) (
Secretory proteins that achieve the correct folded and assembled conformation are then transported from the ER to the Golgi complex in coat protein complex II (COPII) carrier vesicles (
From the
Upon arrival to the plasma membrane secretory proteins are released to the extracellular space by exocytosis. Constitutive exocytosis occurs in all cell types to release extracellular proteins and maintain plasma membrane homeostasis and cell polarity. While regulated exocytosis occurs in specialized secretory cells and is triggered by secretion signals (
Overall the concerted action of many cellular components is required to ensure the selective and efficient secretion of proteins. Many of these steps require energy and mitochondria play a relevant role, fulfilling the ATP requirements of the pathway (
Though the conventional protein secretion pathway was considered for a long time as the only mechanism for protein secretion, we now know that many proteins use alternative pathways. These include the secretion of cytosolic proteins that do not have a signal peptide (leaderless proteins) or a transmembrane domain; and proteins that contain a signal peptide or a transmembrane domain and enter the ER but are not transferred to the Golgi complex (
Proteins without a leader sequence are secreted along three different pathways: Type I pathways involve the translocation from the cytoplasm to the extracellular space through a pore in the plasma membrane. In Type II secretion processes the ATP-binding cassette transporter is responsible for the secretion of the protein present in the cytoplasm while in Type III secretion the proteins use autophagosomes and endosomes to reach the extracellular space. The Golgi-bypassing route is also known as Type IV secretion pathway (
One of the most studied proteins secreted by the unconventional pathways is interleukin-1β (IL-1β) (
Many neurotransmitter molecules are amino acids or amino acid derivatives and their secretion occurs through exocytosis. The pathway starts with the synthesis of the neurotransmitters by enzymes, followed by their loading into of synaptic vesicles. The loading process involves transporters in the vesicle membrane and happens at the expense of an electrochemical proton gradient, generated by the vacuolar H+-ATPase. This pump uses the energy released by ATP hydrolysis to transport protons into the vesicle lumen (
Mitochondria are cell organelles defined by a double membrane, the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM). The OMM separates the mitochondria from the cytosol, however the voltage-dependent anion channel (VDAC or porin) allows the passage of metabolites and ions. In the OMM are also located proteins involved in apoptosis, mitochondrial dynamics, and tethering to other organelles. In the IMM, two functional and structurally different regions are described: the inner boundary membrane and the cristae, where electron transport and ATP synthesis take place in a process known as oxidative phosphorylation (
The ETC complexes are assembled forming supercomplexes that optimize electron transport and proton shuttling through the IMM (
Mitochondria are the main source of ATP in most cells, since many catabolic pathways converge in this organelle and result in the production of ATP by oxidative phosphorylation. The catabolism of metabolites such as glucose, proteins, and fatty acids produces acetyl-CoA, which is in turn oxidized in the tricarboxylic acid cycle generating the reduced electron donors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). Electrons are then transferred to the ETC and flow through the complexes to molecular oxygen (O2). This thermodynamically favorable electron transport releases energy, which is used to pump protons from the matrix to the intermembrane space, at complexes I, III, and IV, creating an electrochemical gradient (
Mitochondrial catabolism of nutrients and electron transport in the respiratory chain involves many redox reactions that have as by-products reactive oxygen species (ROS) (
Under physiological conditions mitochondria produce controlled levels of oxidants, many of which participate in signaling processes. However, ROS formation can increase during cellular stress or in pathological conditions. Although mitochondrial oxidants can be detoxified by enzymatic and non-enzymatic antioxidants (
Mitochondrial dynamics consists of fusion and fission events driven mainly by dynamin-related GTPases (
In turn, mitochondrial fission is carried out by the GTPase dynamin related protein 1 (DRP1), a cytosolic protein that is recruited to the OMM (
Multiple studies linking mitochondrial morphology and bioenergetics can be found in the literature (
In order to face increases in energy demands, such as those imposed by secretion, mitochondria also modify their cellular distribution. Motor proteins, kinesin, and dynein, transport mitochondria over long distances along microtubules toward the cell periphery or the cell center, respectively (
Mitochondria can interact with several cellular compartments, including organelles involved in protein secretion such as the Golgi complex and ER (
Among the proteins found in ERMCs is MFN2, and though some controversy exists (
Physical tethering of mitochondria and the ER by mitofusins (
Calcium ion leaves the ER through the I3PR and enters the mitochondria through VDAC and the mitochondrial calcium uniporter (MCU) complex present in the OMM and IMM, respectively (
In ERMCs we can also find SAR1 and ARF. These small GTPases involved in COPII and COPI vesicle assembly are also required to maintain mitochondrial morphology and function (
Overall, mitochondria are dynamic organelles whose function, morphology, distribution, and contacts with other organelles (
As mentioned before, many of the steps in the conventional secretion pathway demand energy (
The mechanism for ATP transport into the ER in mammalian cells was elusive for many years, and different possibilities, ranging from the existence of specific transporters to non-specific transport through anion channels or leaky membranes, have been proposed (
As we mentioned above, Ca2+ signals for exocytosis in many secretory cells. The increase in cytosolic Ca2+ concentration triggers vesicle fusion with the plasma membrane leading to the release of the cargo to the extracellular space. Calcium enters the cytosol through channels in the plasma or the ER membranes and several processes can affect the magnitude and duration of the signal for exocytosis, among them is Ca2+ uptake by mitochondria (
Ca2+ buffering has different effects on exocytosis, depending on the channel and cell type. Voltage-gated Ca2+ channels (Cav) are present in the plasma membrane. These are activated by membrane depolarization and mediate Ca2+ influx in response to action potentials and other depolarizing signals (
In the plasma membrane we also find store-operated channels, known as Ca2+ release-activated
In the ER membrane the IP3R and ryanodine-sensitive receptors are the channels responsible for Ca2+ release to the cytosol (
From what we just described it is clear that mitochondria contribute to the regulation of intracellular Ca2+ concentrations, and therefore impact on the regulated exocytosis of many proteins and other signaling molecules such as neurotransmitters (
The NLRP3 inflammasome is a multiprotein complex that participates in innate immunity. It is activated by multiple signals of infection, cellular damage, or stress, to produce inflammatory cytokines that trigger innate immune responses (
NLRP3 inflammasome activation occurs in two steps, priming and NLRP3 activation. Priming involves the recognition of pathogen-associated molecular patterns (PAMPs, such as LPS) by pattern recognition receptors or binding of cytokines to receptors. These events lead to the activation of NF-κB and consequent upregulation of NLRP3, caspase 1 and IL-1β and IL-18 gene expression (
Moreover, mitochondria are required for the assembly of the inflammasome. In absence of stimuli the NLRP3 is found in the cytoplasm associated with the ER, while its activation results in an association with mitochondria and enrichment in ERMCs (
Other mitochondrial proteins that are required for NLRP3 inflammasome activation are mitochondrial antiviral signaling proteins (MAVS). These proteins form aggregates in the OMM, associate with NLRP3 and promote its oligomerization, and activation of the inflammasome during RNA virus infections (
In sum, mitochondria provide energy, signaling molecules and a structural scaffold for the assembly and activation of the NLRP3 inflammasome (
Considering the relevant roles of mitochondria in secretion it is not surprising that mitochondrial dysfunction underlies the pathogenesis of certain diseases. Herein we discuss the role of this organelle in some relevant physiological secretion processes, and present evidence linking bioenergetic failure to the development of disease.
Pancreatic β-cells are secretory cells located in the islets of Langerhans in the pancreas. Their main function is to synthesize and secrete insulin, a peptidic hormone responsible for regulating levels of glucose in the blood. Upon the increase in glucose concentrations in plasma, β-cells secrete insulin stored in the secretory granules and increase the synthesis of the hormone (
Mitochondria play a key role in glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells. In these cells glucose is metabolized in the glycolytic pathway and tricarboxylic acid cycle and results in ATP synthesis by oxidative phosphorylation. ATP promotes the closure of ATP-sensitive potassium channels and depolarization of the plasma membrane, leading to the opening of voltage-gated Ca2+ channels. Calcium influx then triggers the exocytosis of insulin granules (
Pancreatic β-cells present an active mitochondrial network where mitochondria constantly undergo fusion and fission events (
It is clear that mitochondria play relevant roles in pancreatic β-cell secretion and a growing body of evidence links mitochondrial dysfunction to impaired insulin release in diabetes mellitus. Diabetes mellitus are a group of metabolic disorders characterized by hyperglycemia, resulting from defects in insulin secretion, insulin sensitivity, or both (
To start with, diabetes mellitus (type 1 and 2) is frequently observed in patients with inherited mitochondrial diseases, which are caused by defects in mtDNA or in nuclear genes encoding mitochondrial proteins that affect mitochondrial ATP synthesis (
Additionally profound changes in mitochondrial metabolism have been observed in pancreatic islets of type 2 diabetes patients and animal models. These include down regulation of components of the tricarboxylic cycle, ETC, ATP synthase and proteins involved in transport across the OMM and IMM (
Neurons are excitable cells that communicate with other cells at synapses. At the chemical synapse neurotransmitters are released by pre-synaptic neurons and bind their receptor at the post-synaptic terminal of target cells. Vesicle discharge at the synaptic cleft occurs in response to an influx of Ca2+ ions through voltage-gated Ca2+ channels, triggered by the action potential. Three different pools of synaptic vesicles can be found in neurons readily releasable pool, the recycling pool, and the reserve pool. The vesicles in the readily releasable pool are released under moderate or intense neuronal activity, while vesicles from the reserve pool are recruited only upon intense stimulation. The latter constitute the majority of vesicles in presynaptic terminals (
Maintaining the electrochemical gradients required for action potentials, transporting, discharging, recycling, and refilling synaptic vesicles, and regulating Ca2+ concentrations are energy demanding processes, that require functional mitochondria (
Due to their high-energy demands, neurons are extremely affected in mitochondrial diseases, caused by mutations in mtDNA, and in other pathologies where mitochondrial function is compromised (
Alterations in energy metabolism can be found in patients and animal models of AD. Reduced glucose metabolism was observed in brain regions affected by AD and the reduction correlated with cognitive decline in AD patients (
Synapse loss is an early event in AD animal models and patients, and exhibits a strong correlation with cognitive deficits (
In this section we present two examples of mitochondrial involvement in secretion processes that underlie pathology. This is not an extensive list, but rather a couple of interesting examples that support the idea that mitochondria might be interesting targets in the design of drugs to modulate secretory processes.
Mast cells are cells of hematopoietic origin that participate in host defense and immunity as well as tissue repair, wound healing, angiogenesis and are also responsible for the development of allergies (
Mast cell degranulation and TNF secretion requires Ca2+ and mitochondrial ATP (
Cellular senescence is triggered in response to stress stimuli. Agents that damage DNA and strong mitogenic signals, such as the expression of oncogenes or loss of tumor suppressors, are strong senescence inducers (
Several studies support that establishment of senescence and the secretory phenotype is accompanied by metabolic reprogramming including increases in mitochondrial oxygen consumption rates, biogenesis and dynamics. In particular, important alterations have been observed in oncogene and therapy induced senescence (
Moreover, mitochondria are required to sustain the SASP and mitochondrial depletion down regulates the expression and secretion of multiple cytokines and other factors in senescent cells (
Since the SASP has been implied in numerous diseases (
Herein we present evidence that mitochondria are required for the successful export of proteins to the extracellular space. Mitochondrial dynamics, bioenergetics and distribution as well as their interactions with other organelles, in particular with the ER, can undergo profound changes in response to secretion stimuli. Mitochondria contribute and support the secretion of proteins providing ATP for energy requiring processes, buffering Ca2+ concentrations and offering structural support and signals for NLRP3 inflammasome activation. A better understanding, at the molecular level, of the role of mitochondria in secretion processes is required and will help in the development of new genetic and pharmacological strategies to modulate protein secretion in pathological contexts.
JM, IM, DT, and CQ contributed to the writing of the manuscript, CQ supervised all the activities. JM made the figures. All authors reviewed contents and approved the final version of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Dr. Adriana Cassina for reading the manuscript and providing insightful comments.
ADP-ribosylation factor
ER chaperone BiP
calcium
coat protein complex I
coat protein complex II
GTPase dynamin related protein 1
ER degradation-enhancing alpha-mannosidase-like proteins
endoplasmic reticulum
endoplasmic reticulum- associated degradation
ER exit sites
ER-Golgi intermediate compartment
ER–mitochondrial contact sites
electron transport chain
fission 1 protein
glucose regulated protein
glucose-stimulated insulin secretion
UDP-glucose:glycoprotein glucosyltransferase
interleukin-1β
inner mitochondrial membrane
inositol-1, 4, 5-trisphosphate
inositol-1, 4, 5-trisphosphate- sensitive channel or receptor
mitochondrial calcium uniporter
mitochondrial contact site and cristae-organizing system
MCU regulator and mitochondrial calcium uptake
mitofusin 1
mitofusin 2
mitochondrial Rho GTPase
Nod-like receptor family, pyrin domain containing 3
outer mitochondrial membrane
optic atrophy protein 1
reactive oxygen species
secretion associated Ras related 1 GTPAse
senescence-associated secretory phenotype
sarcoplasmic/endoplasmic reticulum calcium ATPase
signal recognition particle
tumor necrosis factor, VDAC, voltage-dependent anion channel.
1ERMCs are also known as MERCs or mitochondria-associated ER membranes (MAMs).