Yes, most of gluconeogenesis occurs in the cytosol. The majority of the enzymes that convert non-sugar precursors into glucose are located there. But the full pathway actually spans three cellular compartments: the mitochondria, the cytosol, and the endoplasmic reticulum. Each compartment handles specific steps, and understanding why requires following the carbon as it moves through the cell.
The Three Compartments of Gluconeogenesis
Gluconeogenesis begins in the mitochondria, runs through the cytosol for most of its length, and finishes inside the endoplasmic reticulum. The pathway works this way because certain enzymes are locked into specific compartments and their substrates have to be physically shuttled between them.
The cytosol houses the largest stretch of the pathway. Once carbon arrives there from the mitochondria (more on that below), it travels through a series of reactions that are essentially glycolysis running in reverse. Two key regulatory enzymes, fructose-1,6-bisphosphatase and the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C), both operate in the cytosol. The three rate-limiting, irreversible steps of gluconeogenesis all take place there.
What Happens in the Mitochondria
When the starting material is pyruvate or an amino acid, the pathway must begin inside the mitochondria. Pyruvate carboxylase, the enzyme that converts pyruvate into oxaloacetate, is exclusively a mitochondrial enzyme. This is the first committed step of gluconeogenesis from these substrates, and it cannot happen anywhere else in the cell.
The problem is that oxaloacetate cannot cross the inner mitochondrial membrane on its own. The cell solves this by converting oxaloacetate into a molecule that can cross. In liver cells, mitochondrial malate dehydrogenase reduces oxaloacetate to malate, which then exits the mitochondria through a membrane carrier. Once in the cytosol, malate is oxidized back to oxaloacetate by the cytosolic version of the same enzyme. From there, PEPCK-C converts oxaloacetate to phosphoenolpyruvate, and the cytosolic portion of the pathway continues toward glucose.
There is also an alternative route. Oxaloacetate can be converted to aspartate inside the mitochondria, and aspartate is then transported to the cytosol in exchange for glutamate through a specific carrier protein. Recent research suggests that aspartate may actually be a more suitable transport form than malate for feeding carbons into gluconeogenesis.
Why PEPCK Distribution Matters
PEPCK, the enzyme that converts oxaloacetate to phosphoenolpyruvate, exists in two forms: a cytosolic version (PEPCK-C) and a mitochondrial version (PEPCK-M). The balance between these two forms varies dramatically across species. Human livers split PEPCK activity roughly 50/50 between the cytosol and mitochondria. Chickens, by contrast, concentrate almost all PEPCK activity in the mitochondria.
When PEPCK-M handles the conversion inside the mitochondria, the resulting phosphoenolpyruvate is transported directly to the cytosol, bypassing the need for the malate or aspartate shuttle. Either way, the carbon ends up in the cytosol as phosphoenolpyruvate, ready for the same downstream reactions.
The Final Step Happens in the ER
The very last reaction of gluconeogenesis does not occur in the cytosol. Glucose-6-phosphate, the second-to-last intermediate, is transported from the cytosol into the lumen of the endoplasmic reticulum by a dedicated transporter protein. Inside the ER lumen, glucose-6-phosphatase cleaves off the phosphate group to produce free glucose. This is the final step of both gluconeogenesis and glycogen breakdown.
This compartmentalization serves an important purpose. By keeping glucose-6-phosphatase sealed inside the ER, the cell prevents it from interfering with glycolysis and other cytosolic pathways that depend on phosphorylated sugars. The free glucose produced in the ER lumen is then exported out of the cell and into the bloodstream.
Some Substrates Skip the Mitochondria Entirely
Not every gluconeogenic precursor needs to pass through the mitochondria. Glycerol, released into the blood from fat breakdown, enters the pathway directly in the cytosol. It is phosphorylated and then converted to an intermediate that feeds into the middle of the gluconeogenic pathway, skipping the mitochondrial steps altogether.
Lactate follows a partially cytosolic route as well. Lactate dehydrogenase in the cytosol oxidizes lactate to pyruvate. That pyruvate then enters the mitochondria for carboxylation to oxaloacetate, so the mitochondrial step is still required, but the initial conversion from lactate happens in the cytosol. This is the basis of the Cori cycle, where lactate produced by exercising muscles is shipped to the liver and converted back into glucose.
Where Gluconeogenesis Takes Place in the Body
The liver is the primary gluconeogenic organ, responsible for most of the glucose produced during fasting. The kidneys also contribute meaningfully, with gluconeogenesis occurring specifically in the proximal tubule cells of the kidney cortex. The subcellular compartmentalization of the pathway is the same in both organs: mitochondria, cytosol, and endoplasmic reticulum.
One notable difference is regulation. In the kidney, the gene encoding PEPCK-C responds to changes in blood pH, ramping up gluconeogenesis during metabolic acidosis. The liver version of this gene does not respond to pH shifts. This means the kidneys can increase glucose production under conditions that do not affect liver output, giving the body an additional layer of metabolic flexibility.