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An integral membrane protein is firmly incorporated into a membrane, commonly through contact with the hydrophobic interior of the lipid bilayer. Extraction behavior is an operational distinction: it resists treatments that remove many peripheral membrane proteins. Transmembrane proteins are one class; integral does not mean spanning.^[ref]

A lipid-anchored protein attaches through a covalently linked lipid. The anchor inserts into the lipid bilayer; the polypeptide chain need not cross it. This is a description of attachment rather than a synonym for transmembrane protein.^[ref]

Organelle protein import brings externally synthesized proteins into an organelle. Many plastid proteins made from nuclear genes carry targeting information recognized by machinery that transports them across the envelope membranes. This delivery enables nuclear genes to supply functions inside a plastid, but the presence of targeting information is not itself evidence that a gene was acquired through endosymbiotic gene transfer. ^[ref]

For the endoplasmic reticulum, cotranslational protein translocation illustrates how organelle protein import can begin before a polypeptide chain is complete. Import into the ER lumen and insertion as a transmembrane protein are different outcomes; neither alone establishes subsequent secretion from the biological cell.

A protease protection assay asks whether a barrier prevents an added protease from reaching a protein. A membrane-disruption comparison tests whether protection depends on that barrier.

The membrane-disruption test was performed by David D. Sabatini and Günter Blobel in 1970. Rat-liver microsomes purified by sucrose centrifugation incorporated radioactive leucine during cell-free translation. Samples received cold trypsin and chymotrypsin, with or without membrane-solubilizing detergents. Electron microscopy checked vesicle preservation; size-exclusion chromatography separated protected fragment sizes. Large membrane-associated fragments lost protection after disruption, whereas small ribosome-protected fragments persisted. An exposed ribosome preparation remained digestible when mixed with microsomes, checking that membranes did not simply inhibit the proteases.^[ref]

To read the qualitative diagram, first compare untreated aliquots: they establish that detectable cargo exists before digestion. Next compare external protease with intact versus disrupted membranes. Persistence only while the barrier remains intact supports enclosure, provided the exposed-cargo control confirms active protease in both chemical conditions. The detergent-only aliquot checks that disruption itself does not destroy recovery or detection. Adding membranes after synthesis tests a different alternative: capture of a completed product rather than entry during biological translation.

This is an argument from controlled accessibility. Binding predicts membrane association but generally leaves an exposed cargo digestible. Enclosure predicts protection that depends on membrane integrity. However, protein aggregation, intrinsic resistance to proteolysis, inactive protease or undetected small fragments can obscure this distinction. For a transmembrane protein, expect protection of the enclosed portion rather than necessarily the full chain. Combine fragment size, compartment controls and membrane topology; a protected signal alone does not uniquely specify the whole protein's location.

Protein targeting directs a protein to its cellular destination. Synthesis in the cytosol does not imply that the protein works there.

For entry into the endoplasmic reticulum, distinguish three questions: does the nascent chain reach the membrane, does part of it cross into the ER lumen, and does a segment remain embedded as a transmembrane protein? Cotranslational protein translocation couples these events to biological translation. A signal peptide can initiate delivery without specifying the eventual destination of the mature protein.

This distinction matters experimentally. Finding a protein in a membrane-containing centrifugation pellet could reflect surface binding or protein aggregation. The microsomal protein import experiment instead asks whether an intact membrane prevents an external protease from reaching the product. That tests accessibility across a barrier, rather than proximity to it.

A signal peptide is a short targeting segment of a newly made polypeptide chain. A familiar ER signal sequence lies near the N-terminus and is removed during entry; Günter Blobel and Bernhard Dobberstein's 1975 experiments linked a larger immunoglobulin light chain precursor to a processed product.^[ref]

Cleavage is a separate question from recognition. Not every targeting signal is a cleaved signal peptide: an internal signal anchor can remain part of a transmembrane protein. Conversely, loss of a segment does not by itself prove membrane crossing. Compare the independent size and accessibility readouts in the microsomal protein import experiment.

A transmembrane protein has at least one polypeptide chain crossing the complete lipid bilayer. One crossing is single-pass; several crossings in one chain are multipass. Hydrophobic regions face the membrane interior, while hydrophilic regions can face water.^[ref]