Fusion
The riporter enzyme was fusioned or inserted to a given point of the transmembrane protein investigated. The activity of the riporter enzyme in this chimera shows the localisation of fusion point.
| PhoA | Fusion with alkaline phosphatase. Alkaline phosphatase is a periplasmic bacterial enzyme. It is active only in the periplasmic space. |
| PhoAS | Sandwich fusion with alkaline phosphatase. Alkaline phosphatase is inserted into a given point of the transmembrane protein investigated. In this case the transmembrane protein is not truncated. Alkaline phosphatase is active only the periplasmic space of bacterii. |
| LacZ | Fusion with beta-galactosidase. Beta-galactosidase is a bacterial enzyme, which is active only in the cytoplasm. |
| PhoALacZ | Fusion with alkaline-phosphatase and beta-galactosidase. Because these enzymes are active on the opposide side of the bacterial inner membrane, only one of them can be active in a given construct. |
| BlaM | Fusion with beta-lactamase. Beta-lactamase is a bacterial enzyme, which protects cells against lysis by beta-lactam antibiotics such as ampicillin. It is active only in the periplasmic space. |
| BAD | Fusion with biotin acceptor domain. Membrane topology studies based on the BAD make use of the compartment-specific in vivo biotinylation of the domain or the high sensitivity to detect the biotinylated proteins in combination with proteolysis. |
| PL | Prolactin fusion. The construct can be digested with PNGase if the fusion position is in the cytoplasm. If digestion occured, the fragment(s) can be detected by gel electroforesis. |
| GFP | Green fluorescent protein fusion. It is possible to fuse GFP to the C-terminal of the investigated protein, or fusing the investigated protein to GFP (prot-GFP or GFP-prot). |
| HIS | Fusion with invertase plus histidinol dehydrogenase (invHIS4C), which is active only at the cytoplasmic site. |
| SplitUbiquitin | The system utilizes complementation between separable domains of ubiquitin: the N terminus of ubiquitin (Nub, amino acids 1-34) and the C terminus of ubiquitin (Cub, amino acids 35-76), which is followed by a reporter protein (Rep). Wild-type Nub (NubI, with I being isoleucine at position 13) spontaneously assembles with Cub-Rep, resulting in proteolytic cleavage at the C terminus of Cub by a ubiquitin-specific protease(s) and subsequent release of the reporter fragment. However a mutant of Nub (NubG) in which Ile-13 is changed to Gly-13 is unable to assemble with Cub-Rep unless two proteins X and Y that interact with each other are fused to the Cub-R and to the NubG so that this interaction can force the reassociation of the two halves of ubiquitin. As a result, the interaction between two proteins X and Y can be monitored by the cleavage and subsequent activation of the reporter genes. |
| Suc2 | Fusion with Suc2p invertase. Suc2p becomes rapidly modified by asparagine-linked glycosylation at multiple sites upon translocation to the lumen of the ER, resulting in a 20-26-kDa increase in molecular mass. |
| Other | Other fusions. |
| HA-Suc2-His4C | Fusion with hemagglutin, Suc2p invertase and histidinol dehydrogenase. |
| APEX | APEX techniques are proximity labelling methods based on fusion to an aspecific peroxidase creating free radicals. This can trigger covalent crosslinking of labelling agents to nearby, topologically matching segments, and also sufficiently alters the proteins in the compartment to make them directly visible under electron microscopy. |
| SplitGFP | The Split-GFP techniques are specialized split-protein assays relying on creation of a chromophore in the matching compartment. It is possible to complement either a GFP fragment-tagged TM protein with the matching soluble GFP fragment, or trigger complementation between two membrane proteins if both their fused tags are oriented into a matching compartment. |
| SpyTag | The SpyCatcher/SpyTag technique rely on a special protein domain (of bacterial origin) that can autocatalytically install covalent crosslinks into itself, or link two halves (SpyTag and SpyCatcher) of the same domain after folding together. This yields a split-protein assay where the irreversible crosslinks (to a chromophore-fused SpyCatcher protein) can be monitored by washing away non-crosslinked proteins. |
| Ubiquitin-GFP | This assay relies on deubiqutylase-mediated removal of linear ubiquitin segments out of any protein. Deubiquitylating enzymes are only present in the cytoplasmic compartment, and can cleave off the chromophore-tagged ubiquitin from the TM protein. |
PostTransMod
Post translational modification: glycolysation or phosphorylation.
| NGlyc | Transmembrane proteins are glycolysated in the endoplasmic reticulum at specific sequence motifs, if the motif is outside. Inserting or deleting glycolysation specific sequence motif, and investigating the molecule mass of the modified protein can help to localise the place of the insertion point. Wild-type N-glycolysation site can be proven to be glycosylated, usually with inhibition of glycolysation and molecular weight shift, or with mutation elimiminating the glycolisation site. Glycosylation of the domain indicates a luminal position of the fusion point while no glycosylation indicates a cytoplasmic location. |
| Cman | C-Mannosylation is a unique form of protein glycosylations, involving the C-glycosidic attachment of a mannosyl residue to the indole moiety of Trp. |
| Phosphorylation | Protein phosphorylation can happen both in internal as well as extracellular compartments. Nevertheless, if the perpetrating kinase, with a known localization is also identified (e.g. by knockout, mutation or inhibitor assays), then phosphorylation can also provide valid toplological information. |
| Ubiquitination | The conjugation of ubiquitin onto target proteins can only occur in the internal (cytoplasmic or nuclear) compartment |
| Cleavage | Proteolytic cleavages can happen either intra- or extracellularly. However, if the perpetrator protease has a known localization, then these modifications are also proof of topology. |
| Lipidation | Lipidation of proteins is performed by enzymes either in the cytoplasm, or in the membrane with a catalytic site facing intracellularly. Therefore most of the known modifications (prenylation, myrystoylation, palmitoylation, etc.) are seen on ether intracellular segments or intramembrane residues directly adjacent to cytoplasm. |
| Other glycans | Glycosylation of proteins happens both intra- and extracellularly, but some types of glycans are highly specific to either compartment. |
| Nmyri | N-mirystoylation of proteins at a properly processed N-terminus is performed by cytoplasm-facing enzymes. Therefore the modified positions attach to the membrane exclusively from the inner side. |
Protease
Membrane proteins, like all proteins, contain cleavage sites for various proteolytic enzymes. Externally added proteolytic enzymes cannot cross the membrane, and therefore cytoplasmic sites are protected against cleavage by the membrane upon exposure of bacterial right-side-out membranes or spheroplasts whereas these sites are not protected against cleavage in inside-out membranes.
| Partial Proteolysis | Digestion with a proteinase enzyme (proper name is given in the value tag) and determining the molecular weight shift. Proper name of the protease is given in the Value field of the database entry. The abbreviations used are: ProtK, Proteinase K; ProtKEP, Proteinase K digestion followed by epitop detection; Tryp, Trypsin; ChyT, Chymotrypsin; V8, Endopeptidase Glu-C (V8 peptidase); ArgE, Arginin-C endopeptidase; AmpK, Aminoipeptidase K; AmpM, Aminopeptidase M; Amp, Aminopeptidase; Subs, Substilisine; CarbA, Carboxipeptidase A; CarbY, Carboxypeptidase Y; Kall, Kallikrein; ThLy, Thermolysin; GlnC, Endopeptidase Gln-C; Papa, Papain; LysC, Lys-C endopetidase; Peps, Pepsin; Clos, Clostripain; Lys-X, Endopeptidase Lys-X; Arg-X, Endopeptidase Arg-X. |
| Signal Peptidase | Signal peptidase enzyme on a N-terminal labelled protein can be used to localise the signal cleveage site. |
| TID | Labeling membrane embedded parts of a protein with 3-(trifluoromethyl)-3-(m-[125] iodophenyl)diazirine ([125I]TID) followed by proteolysis. The peptides attached to the membrane remained labeled and can be identified. |
Immunolocalisation
Localisation of inserted epitope using specific antibody. Proper name of the protease is given in the Value field of the database entry. The abbreviations used are: FMDV, Foot and mouth disease virus; FETfl, Functional Epitope Tagging of Flag; FETcm, Functional Epitope Tagging of c-myc; HAEI, Hemagglutinin; ColA, Colicin A; PP, 6-4 Photoproduct.
| Epitope Insertion | Inserting artificial epitope for immunolocalisation. |
| Endogen Epitope | Using monoclonal antibody against the endogen epitope of proteins. |
Chemical_modification
Chemical modification of amino acid side chains.
| Cys | The cysteine residue is a relatively hydrophobic, nonbulky residue, and its introduction at most positions in a membrane protein is likely to be tolerated. This feature and the ease of specific chemical modification with sulfhydryl reagents are the basis of several methods aiming at topological and structure-function-related information on membrane proteins. In cysteine scanning mutagenesis, a series of single cysteine mutants created and the reactivity of the single cysteine mutant to various sulfhydryl reagents is assessed under different conditions. |
| Lys | Chemical modification of lysin by an activated, membrane impermeable carboxyl acid, like Sulfo-NHS-Biotin. |
| COOH | Chemical modification of carboxyl groups by a two step reaction (activation and labeling). |
| Quenching | Fluorescence quenching using PM-labelled (N-(1-pyrenyl)-maleimide) single-Cys mutants, water-soluble (e.g. acrylamide) and lipid-soluble (e.g. 5-doxylstearic acid, 12-doxylstearic acid) quenchers. |
Structure
Extracting topology data directly from the 3D structure of the protein.
| PDBTM | 3D Structure from PDB processed by the TMDET algorithm. Side definitions are defined using original publications. |
| PDB | 3D Structure from PDB. Side definitions are defined using original publications. |
Other
Other techniques not classified above.
| Revertants | Revertants |
| SeqMotif | Sequence motif, which always is specific to one side of the membrane. The Value field contains the name of the motif (currently only Walker A or B, ABC Signature, ATP-Binding, Transit and OGlyc). |
| Tailoring | Deletion or insertion some residues or segments and investigating the properties of the protein. The name of the method is on the Value field. It can be: TCSI Trypsin Cleavage Site Insertion: 31 aa long insertion containing a lot of trypsin cleavage site (PMID:10498725). fXa Factor Xa protease cleavage site insertion. DPerm Deletion are in permissive site, i.e. properties (function, structure) are not affected by the deletion indicating surface loops. DTM Deleting transmembrane region or regions. M0M1 The potential transmembrane segments are inserted to fusion vectors to find signal anchor and/or stop transfer sequences. DTMNGlyc Glycosylation altered by upstream transmembrane region deletion DTMPhoA PhoA fusion activity altered by upstream transmembrane region deletion |
| Annotated linear motif | In case of an experimentally established protein-protein interaction though a well-defined motif, the motif must lie in the same compartment as its partner for the interaction to be observable. |
| Spin labelling EPR | This protein structural assay allows the determination of the exact tilt angles at which particular segments (including TM segments) are located at. |