PSORT Users' Manual

Introduction

Input Information

Source of Input Sequence

Select one of the radio buttons to specify the source origin of the input sequence. This selection determines the candidate localization-sites for prediction as listed below:

Gram-positive bacterium:

(cytoplasmic) membrane, cytoplasm, and outside, i.e., the protein will be secreted.

Gram-negative bacterium:

cytoplasm, inner membrane, periplasm, and outer membrane.

yeast:

cytoplasm, mitochondria (outer membrane, intermembrane space, inner membrane, and matrix space), microbody (peroxisome), nucleus, endoplasmic reticulum, abbreviated as ER, (lumen and membrane), Golgi body, vacuole, plasma membrane, and outside.

animal:

cytoplasm, mitochondria (outer membrane, intermembrane space, inner membrane, and matrix space), microbody (peroxisome), nucleus, endoplasmic reticulum (lumen and membrane), Golgi body, lysosome (lumen and membrane), plasma membrane, and outside.

plant:

cytoplasm, mitochondria (outer membrane, intermembrane space, inner membrane, and matrix space), microbody (peroxisome), nucleus, endoplasmic reticulum (lumen and membrane), Golgi body, vacuole, plasma membrane, outside, and chloroplast (stroma, thylakoid membrane, and thylakoid space).

Sequence ID

Enter the sequence here by direct typing or by the copy & paste feature of any window systems. Characters except standard one-letter code for 20 amino acids, e.g., spaces, numeric, and carriage returns, will be removed off by the system. Small cases will be changed to capital cases. The input sequence is expected to be a direct translation from the genetic information and to contain all information for sorting. Thus, a warning message will be issued if it starts by an amino acid except M (methionine).

Output for Bacterial Sequences

In Gram-negative bacteria, most periplasmic and outer membrane proteins have a signal sequence (also called a leader peptide) in the N-terminus, which is cleaved off after the translocation of the cytoplasmic membrane. Some of the cytoplasmic membrane proteins also have cleavable signal sequences but some N-terminal signal sequences in the cytoplasmic membrane proteins are not cleaved off, remaining as transmembrane segments.

In general, hydrophobic transmembrane segments exist in the cytoplasmic membrane proteins only. Thus, these segments can be regarded as the sorting signal into the cytoplasmic membrane.

The signal sequence of lipoproteins, i.e., proteins with a covalently attached lipid molecule in their mature N-terminus, are essentially the same as those of usual proteins except the region around their cleavage sites. Thus, they can be recognized by the combination of McGeoch's method and the consensus motif around the cleavage site formulated by von Heijne (G. von Heijne, Protein Eng., 2, 531, 1989). The program is named as "Lipop" here. It gives the possible modification site around the end position of preceding CR region defined in McGeoch's method for a probable lipoprotein; otherwise, it returns a dummy modification site, -1.

Output for Eukaryotic Information

In eukaryotes, proteins sorted through the so-called vesicular pathway (bulk flow) usually have a signal sequence (also called a leader peptide) in the N- terminus, which is cleaved off after the translocation through the ER membrane. Some N-terminal signal sequences are not cleaved off, remaining as transmembrane segments but it does not mean these proteins are retained in the ER; they can be further sorted included in vesicles.

The current version of PSORT assumes that all integral membrane proteins have hydrophobic transmembrane segment(s) which are thought to be alpha- helices in membranes.

PSORT employs Klein et al.'s method ("ALOM", also called as KKD) to detect potential transmembrane segments (P. Klein, M. Kanehisa, and C. DeLisi, Biochim. Biophys. Acta, 815, 468, 1985). It attempts to identify the most probable transmembrane segment from the average hydrophobicity value of 17-residue segments, if any. It predicts whether the segment is a transmembrane segment (INTEGRAL) or not (PERIPHERAL) comparing the discriminant score (reported as 'value') with a threshold parameter pre- defined to 0.0 for bacteria ('threshold'). For an integral membrane protein, position(s) of transmembrane segment(s) are also reported. Their length is fixed to 17 but their extension, i.e., the maximal range that satisfies the discriminant criterion, is also given in parentheses. The discrimination step mentioned above is continued after leaving out the segment till there remains no predicted transmembrane segment. The item 'count' is the number of predicted transmembrane segments.

Membrane proteins have their spcefic way to integrate into the membrane in respect to the two sides (cytoplasmic or exo-cytoplasmic), which is called as membrane toplogy. We used Singer's classification for membrane topology (S. J. Singer, Ann. Rev. Cell Biol., 6, 247, 1990). Prediction of membrane topology is important because some sorting signals reside in specific positions in specific topologies, e.g., cytoplasmic tail (see below).

PSORT uses Hartmann et al.'s method (E. Hartmann, T. A. Rapoport, and H. F. Lodish, Proc. Natl. Acad. Sci. USA, 86, 5786, 1989); called "MTOP" here) for the prediction of membrane topology, which assumes that the overall topology is determined from the net charge difference of both sides of 15 residues flanking the most N-terminal transmembrane segment. In the outpu, 'I(middle)' means the central position of the most N-terminal segment.

Since the N-terminal transmembrane segments of type Ib proteins were often wrongly predicted to be cleaved off by von Heijne's method, we introduced the hypothesis that if the charge difference of the most N-terminal transmembrane segment is reversed to that of usual ER-transferons, it is not cleaved. Since some cleavable ER-transferons had a reversed charge difference, we had to change the originally reported threshold value. PSORT also uses a heuristic that transmembrane segments of many type II proteins reside apart from the N-terminus to some degree.

In mitochondria, many proteins are sorted through a 'conservative' pathway while others are sorted through 'nonconservative' pathways from the cytoplasm. The proteins sorted through the former have mitochondrial matrix targeting signals in their N-terminus. On the contrary, sequence features of protein sorting signals with 'nonconservative' pathways are hardly recognizable.

Although it seems possible that a protein without its own nuclear targeting signal enters the nucleus via cotransport with a protein that has one, many nuclear proteins have their own targeting signals. Their most common type is that of SV40 large T antigen. PSORT uses the following two rules to detect it: 4 residue pattern composed of basic amino acids (K or R), or composed of three basic amino acids (K or R) and H or P; a pattern starting with P and followed within 3 residues by a basic segment containing 3 K or R residues out of 4 residues.

Another type of nuclear targeting signal is the type of Xenopus nucleoplasmin proposed by Robbins et al. (J. Robbins, S. M. Dilworth, R. A. Laskey, and C. Dingwall, Cell, 64, 615, 1991). The pattern is: 2 basic residues, 10 residue spacer, and another basic region consisting of at least 3 basic residues out of 5 residues.

PSORT used a heuristic that nuclear proteins are generally rich in basic residues: If the sum of K and R compositions are higher than 20%, then the protein is considered to have higher possibility of being nuclear than cytoplasmic. In addition, it also examines the presence of RNP (ribonucleoprotein) consensus motif because some RNPs are transported to the nucleus by signals existing in the bound RNAs. However, it is apparently insufficient for actual prediction.

Peroxisomes, sometimes called glyoxisomes, glycosomesare, or microbodies, are organelles found in almost every eukaryotic cell. As a sorting signal, the importance of the C-terminal three residues, (S/A(/C))(K/R/H)L, has been indicated (the SKL motif). However, since many peroxisomal proteins do not have this motif at the appropriate position, PSORT uses a heuristic that the presence of this motif at other positions also implicates the peroxisomal localization.

Although some peroxisomal proteins have N-terminal presequences which are cleaved off after translocation, it is not clear whether they are sorting signals. According to our preliminary analysis, the amino acid composition of the N-terminal 20 residues were not very effective as variables of discriminant analysis. Then, the amino acid composition of the entire sequence is used for supplemental information for prediction.

PSORT postulates that the proteins with N-terminal signal sequence will be transported to the cell surface by default unless they have any other signals for specific retention or commitment; a luminal protein will be secreted constitutively to the extracellular space and a membrane protein will reside at the plasma membrane.

The retention signal of ER luminal proteins from the bulk flow is the existence of the sequence motif KDEL in the C-terminus. In yeast and some plants, the consensus motif is HDEL. Although some variations of this motif are allowed in some organisms and cell types, they were not required for the discrimination of our current data.

As already exemplified above, many sorting signals in the membrane proteins have been found in cytoplasmic tails which are short terminal segments exposed to the cytoplasm in type Ia, Ib, and II proteins (in Singer's terminology).

In relation to the default pathway of secretion, there is a pathway for protein internalization through coated-pit mediated endocytosis. Two sequence motifs, NPXY and YXRF, have been identified as signals for this rapid internalization process. PSORT uses these sequence motifs as clues identifying plasma membrane proteins.

The protein modification reactions which bind lipid molecules to proteins are important because a linked lipid moiety can be integrated into various membranes and can anchor the bound protein.

For example, myristoylations occur at the consensus sequence in the N-terminal 9 residues. However, recent studies suggest that many of them may not take part in the direct anchoring. Thus, PSORT does not use the result for further reasoning although the observation will be reported.

In contrast, all proteins linked to the glycosyl-phosphatidylinositol (GPI) molecules are thought to be anchored at the extracellular surface of the plasma membrane. PSORT recognizes GPI-anchored proteins by the knowledge that most of them are predicted to be type Ia membrane proteins with very short cytoplasmic tail (within 10 residues) and uses the result for the prediction of the localization site (plasma membrane) of the modified protein.

Lysosomes are acidic organelles that contain numerous hydrolytic enzymes. In yeast and plant cells, similar functions are recognized in vacuoles, which have diverse functions.

For soluble lysosomal proteins, the pathway which utilizes the post- translational modification of mannose 6-phosphate has been clarified. However, there are no clear consensus patterns except for the NX(S/T) pattern necessary for N-glycosylation, likely because the modification is conformation-dependent. Since the prediction of protein conformation is very difficult, PSORT uses the discriminant score based on amino acid composition.

It is likely that yeast and most plant cells share part of their sorting mechanism. Many of them have signal sequences in their N-terminus and have pro regions that are cleaved off after translocation. Nevertheless, no common sequence features have been observed. Again, PSORT uses the information of amino acid composition for discrimination The amino acid composition of lysosomal and vacuolar soluble proteins turned out to be totally different.

The sorting mechanism of lysosomal membrane proteins seems different from that of lysosomal luminal proteins. The existence of a GY motif within 17 residues from the membrane boundary in the cytoplasmic tails of type Ia proteins is used as a rule for discrimination.

Notes on the Knowledge-based System