Organelles as Distinct Environments

In cultured HeLa cells, the inside of the mitochondrion measured close to pH 8.0 and the Golgi close to pH 6.6. In mammalian cells the lysosome sits at pH 4.5 to 5.0. All three share one cytosol.

That spread is larger than it looks, because pH is a logarithm. A cell keeps compartments that differ a thousandfold or more in free protons, and others that differ several thousandfold in free calcium, side by side. This lesson asks how it does that, what each compartment keeps, how you would find out where an unknown protein lives, and why two of these compartments carry their own DNA.

The idea: membranes let a cell run several chemistries at once

Think of a building in which every room has its own thermostat. The walls do not make a room warm. They slow the loss of heat, so a heater can hold the room at a set point. An organelle works the same way. Its lumen, the space enclosed by its membrane, is kept different from the cytosol, the fluid part of the cell outside all organelles, by pumps in the membrane working against leak.

The wall here is the lipid bilayer, which ions cross only slowly unless a channel or carrier lets them through (see the lesson on membrane transport). Every luminal difference you meet below is therefore a steady state. It lasts only as long as a pump keeps paying, usually in ATP. Stop the pump and the difference runs down.

Why bother? Because different jobs need different chemistry. Lysosomal hydrolases such as the protease cathepsin D work best in acid. Disulfide bonds form in an oxidizing lumen. A calcium store is useful only if its calcium is far above the cytosol’s, so that opening a channel sends a signal (that release is covered in the lesson on calcium signalling). And ATP synthesis in the mitochondrion depends on a proton gradient across the inner membrane (see oxidative phosphorylation).

A chemical map of the cell

Here is what has been measured. Each value keeps its source, because the map combines single-cell measurements with review ranges.

Compartment	Value	System
Nucleus	pH near 7.2	Mammalian cells, review
ER lumen	pH near neutral	HeLa cells, targeted reporter
Medial/trans Golgi	pH 6.58	HeLa cells, targeted reporter
Early and recycling endosomes	pH about 6.3 to 6.5	Mammalian cells, review
Late endosomes	pH about 5.5	Mammalian cells, review
Lysosomes	pH 4.5 to 5.0; Ca2+ about 0.5 mM; Cl− 80 to 100 mM	Mammalian cells, review
Mitochondrial matrix	pH about 7.98 (estimated)	HeLa cells, targeted reporter
ER lumen	free Ca2+ about 0.5 to 1 mM	Mammalian cells, review
Cytosol	free Ca2+ about 100 nM at rest	Mammalian cells, review
Chloroplast stroma, in light	pH about 7.8 to 8.0	Older plant measurements, reviewed

The nucleus shares much of the cytosol’s ionic chemistry, because its envelope is perforated by pores through which small ions pass freely.

Two traps sit in this table. First, not every organelle is acidic. The ER is near neutral, and the matrix is alkaline. Second, oxidizing does not mean acidic. In cultured CHO and HCT116 cells, a redox reporter called CY-RL7 was about 88% oxidized in the ER lumen and fully reduced in the cytosol, while the ER’s pH stays near neutral. Redox describes electrons; pH describes protons.

Redox also depends on which chemical couple you measure. In budding yeast, a reporter coupled to glutathione estimated the cytosol at about −306 mV and the matrix at about −301 mV, so the matrix pool was strongly reducing even though mitochondria make reactive oxygen species. In the same yeast study, the glutathione pool of the intermembrane space exchanged with the cytosol through porins, but no appreciable exchange with the matrix was detected. In mammalian cultured cells, the redox state of the peroxisome depended strongly on growth conditions. No reliable peroxisomal pH is given here, so none is stated.

Worked problem: reading the map and placing a protein

Part 1. How many more free protons are in the Golgi than in the matrix? Use the two HeLa means, 6.58 for the Golgi and 7.98 for the matrix. They come from separate measurements, not one cell read twice.

pH = −log10(aH+), so aH+(Golgi) / aH+(matrix) = 10^(pH matrix − pH Golgi)

1. Subtract: 7.98 − 6.58 = 1.40.
2. Raise ten to that power: 10^1.40 = 25.1.
3. Check by computing each side, treating activity as concentration: 10^−6.58 = 2.63 × 10^−7 M (0.263 µM) and 10^−7.98 = 1.05 × 10^−8 M (0.0105 µM). Then 0.263 / 0.0105 = 25.1.

The Golgi lumen has about 25 times the free proton activity of the matrix. This is free activity only. It says nothing about the total protons held by buffers or how fast protons move.

Compartment 1 Compartment 2

add bafilomycin A1 (V-ATPase inhibitor), HeLa Golgi only

Each pH unit is a tenfold change in free protons, so the ratio is 10 raised to the pH difference. It compares free proton activity only; it says nothing about the total protons held by buffers or how fast they move. This is a sketch of the logic, and the values come from different systems.

Part 2. Which compartment holds protein X? Suppose a lab breaks cells and separates them into four fractions, F1 to F4, and measures each protein’s share in each fraction. These numbers are made up for practice. The three markers are ones used in a real HeLa map.

Protein	F1	F2	F3	F4
Histone H3 (nucleus)	0.80	0.10	0.05	0.05
Cathepsin D (lysosome)	0.05	0.20	0.60	0.15
Pyruvate kinase (cytosol)	0.05	0.05	0.10	0.80
Unknown X	0.06	0.18	0.61	0.15

1. Compare whole shapes, not one peak. X differs from cathepsin D by 0.01, 0.02, 0.01 and 0.00, a total of 0.04.
2. Against histone H3 the differences are 0.74, 0.08, 0.56 and 0.10, a total of 1.48.
3. Against pyruvate kinase they are 0.01, 0.13, 0.51 and 0.65, a total of 1.30.

X is provisionally lysosomal. Before concluding, you would want a second lysosomal marker that matches, replicate fractionations and an imaging check in intact cells. Large studies leave some proteins unclassified because they live in more than one compartment (for the logic of markers, see the lesson on experiments that distinguish).

How pumps keep the differences

The V-ATPase is a rotary pump that spends ATP to move protons from the cytosol into a lumen. A mammalian review names it the main mechanism keeping endosomes and lysosomes acid. In HeLa cells, the V-ATPase inhibitor bafilomycin A1 made the Golgi less acid, which shows that Golgi acidity is actively kept. Pumping protons alone would also build up positive charge in the lumen and slow the pump, so acidification is thought to need a counterion: chloride moving in, or another cation moving out. Why each compartment along the route from early endosome to lysosome settles at a different pH, with one pump family acidifying them all, is still studied.

SERCA, the sarco/endoplasmic reticulum Ca2+-ATPase, pumps calcium from the cytosol into the ER and builds the gradient across the ER membrane. From the table, 0.5 to 1 mM against 100 nM is a 5,000- to 10,000-fold difference. Block SERCA with thapsigargin and calcium leaks out of the ER faster than it is replaced.

Mitochondria and chloroplasts run the pumps in other directions. The respiratory chain pumps protons out of the matrix, which is why the matrix is alkaline (see the proton-motive force). In chloroplasts, light drives protons into the thylakoid lumen and the stroma becomes alkaline. The proton-motive force there includes both a pH difference and a voltage (see the light reactions).

A pump that sets one gradient need not set another. In one HEK293 assay, the lysosome’s calcium store refilled after release even with the V-ATPase blocked, but failed to refill when ER calcium was depleted or chelated. That result holds for that cell line and assay, and the transporter that takes calcium up was not identified. The lysosome also serves as a platform for nutrient signals, which the lesson on mTOR and AMPK covers.

Bacteria inside: the endosymbiotic evidence

Mitochondria and plastids differ from most other organelles in having their own DNA and ribosomes, inside two envelope membranes. The idea that they descend from bacteria taken into a host cell was proposed for plastids by Konstantin Mereschkowski early in the twentieth century. In 1967, writing as Lynn Sagan, Lynn Margulis assembled the case for both organelles. Her further proposal that eukaryotic flagella came from spirochetes has not been supported.

Molecular sequences tested the idea. Trees of mitochondrial ribosomal RNA placed mitochondrial sequences within the Bacteria rather than with archaea or the eukaryotic cytosol, and later protein data pointed to the Alphaproteobacteria. A 2022 analysis of 108 mitochondrial proteins, using a model that allows for changes in amino-acid composition across sites and branches, favoured mitochondria as sister to the Alphaproteobacteria, excluding two related lineages (MarineProteo1 and Magnetococcia), rather than inside any living group. For plastids, phylogenomic analyses supported a cyanobacterial origin, and in 2017 the closest cultured relative was Gloeomargarita lithophora. That species is a cousin of the plastid line, not its ancestor.

The genomes tell the rest. Much of the ancestral mitochondrial genome moved to the nucleus by endosymbiotic gene transfer, so most mitochondrial proteins are now made in the cytosol and imported (see protein sorting). The modern mitochondrial proteome is a mosaic, with many proteins of origins outside the Alphaproteobacteria. Nor are mitochondrial genomes all circles: that of Proteromonas lacertae is linear. Peroxisomes, by contrast, have no genome; they share the work of breaking down fatty acids with mitochondria (see fatty acid degradation).

Design problem

You suspect that a protein lives in the lysosome lumen and needs low pH to work. Design experiments to test both parts.

One way to do it

Start by confirming the environment. Express a pH reporter targeted to lysosomes, calibrate it in the cells with ionophores, and check that it reads in the expected acid range for your cell type.

Next, place the protein. Fractionate the cells and compare its profile with at least two lysosomal markers, such as cathepsin D, and with markers of nearby compartments. Test latency: if the protein is inside the lumen, its activity on an external substrate should rise when membranes are opened with detergent. Confirm by imaging against a lysosomal marker in intact cells.

Then test the pH requirement. Add bafilomycin A1 to block the V-ATPase, and measure both the rise in lysosomal pH and the protein’s activity over time. Include a control showing that bafilomycin has not simply stopped the protein from reaching lysosomes, for example that its amount in the lysosomal fraction is unchanged. A stronger test measures the purified protein’s activity across a pH range in vitro.

Keep the language of evidence. Activity that falls as pH rises is a correlation; the in vitro pH curve and a rescue (restoring activity by restoring acidity) are what argue for cause.