The EMBO Journal vol.12 no.6 pp.2575-2583, 1993
Protein splicing of the yeast TFP1 intervening protein
sequence: a model for self-excision
Antony A.Cooper, Yen-Ju Chen,
Margaret A.Lindorfer1 and Tom H.Stevens2
Institute of Molecular Biology, University of Oregon, Eugene,
OR 97403, USA
'Present address: Department of Pathology, University of Virginia
Health Science Center, Charlottesville, VA 22908, USA
Communicated by D.Meyer
Protein splicing is the protein analogue ofRNA splicing
in which the central portion (spacer) of a protein
precursor is excised and the amino- and carboxy-terminal
portions of the precursor reconnected. The yeast Tfpl
protein undergoes a rapid protein splicing reaction to
yield a spliced 69 kDa polypeptide and an excised 50 kDa
spacer protein. We have demonstrated that the 69 kDa
species arises by reformation ofa bona fide peptide bond.
Deletion analyses indicate that only sequences in the
central spacer protein of the Tfpl precursor are critical
for the protein splicing reaction. A fusion protein in which
only the Tfpl spacer domain was inserted into an
unrelated protein also underwent efficient splicing,
demonstrating that all of the information required for
protein splicing resides within the spacer domain.
Alteration of Tfplp splice junction residues blocked or
kinetically impaired protein splicing. A protein splicing
model is presented in which asparagine rearrangement
initiates the self-excision of the spacer protein from the
Tfpl precursor. The Tfpl spacer protein belongs to a
new class ofintervening sequences that are excised at the
protein rather than the RNA level.
Key words: mobile genetic element/protein introns/protein
Protein splicing is one of many processes that modify the
informational flow from gene to mature protein. This unusual
process is exemplified by the Saccharomyces cerevisiae
TFPI gene product (Kane et al., 1990). TFPI encodes a
119 kDa protein (Tfplp) that undergoes protein splicing to
produce both the 69 kDa catalytic subunit of the vacuolar
H+-ATPase and a 50 kDa spacer protein (Shih et al., 1988;
Hirata et al., 1990; Kane et al., 1990; Hendrix, 1991; Hirata
and Anraku, 1992). This reaction involves the excision of
the intervening spacer protein from the central portion of
the 119 kDa precursor protein, and the joining of the N-
and C-domains to form the vacuolar H+-ATPase subunit
(Figure 1). Protein splicing ofTfplp has been shown to occur
in Escherichia coli, yeast, and when translated in vitro (Kane
et al., 1990).
Two additional examples ofprotein splicing have recently
been discovered: RecA from Mycobacterium tuberculosis
(Davis et al., 1991, 1992) and DNA polymerase from the
(Hodges et al., 1992; Perler et al., 1992). In each case an
intervening amino acid sequence, with homology to the
spacer protein ofTfpIp, separates two domains ofthe mature
protein (Shub and Goodrich-Blair, 1992). Both the RecA
and DNA polymerase spacer sequences have been shown
to be removed post-translationally, and genetic evidence
suggests that the majority of the N- and C-domains of the
RecA precursor are not required for the splicing reaction
(Davis et al., 1992; Hodges et al., 1992).
A seemingly less related post-translational polypeptide
rearrangement occurs in the maturation of the plant lectin
concanavalin A (Carrington et al., 1985). The process
involves the cleavage and formation of peptide bonds, yet
it is different from the above cases of protein splicing. For
concanavalin A, the reaction results in reversing the order
of the precursor's N- and C-domains rather than excising
a large intervening protein sequence (Bowles et al., 1986;
Bowles and Pappin, 1988).
The excised Tfplp spacer protein has recently been identi-
fied as a highly specific DNA endonuclease that cleaves a
site in a TFPI allele that is created by the exact deletion of
the spacer DNA (TFP1-spacerA allele; Bremer et al., 1992;
Gimble and Thomer, 1992). In vitro studies with the purified
spacer protein [designated VDE by Gimble and Thorner
(1992)] demonstrated that cleavage occurred within the
TFPI-spacerA DNA at the N/C domain junction, but the
spacer protein did not cleave the wild-type TFPI DNA.
Cleavage of the TFPJ-spacerA gene by the spacer protein
in a TFP1ITFP1-spacerA heterozygote was shown to initiate
gene conversion that converted an allele that lacked the inter-
vening sequence (TFPI-spacerA) into one that contained it
(TFP1). These observations demonstrated that the spacer
protein is capable ofmediating the movement of its encoding
DNA sequence, thereby indicating that the TFP1 intervening
sequence is genetically mobile.
We investigated the mechanism of protein splicing and
report here that the spacer protein can splice from a
completely unrelated insertional context. Mutational analysis
of the residues at the Tfplp splice junctions reveals that
certain residues are critically important for the protein
splicing reaction. To account for our findings, we propose
a protein splicing model involving self-excision ofthe spacer
Protein splicing joins the N- and C-domains via a
Previous experiments have indicated that the TFPI-encoded
119 kDa precursor (Tfplp) undergoes protein splicing to
produce the 69 kDa vacuolar H+-ATPase subunit (Kane
© Oxford University Press
Fig. 1. Protein splicing of yeast Tfplp. The schematic diagram shows
the 119 kDa Tfplp precursor protein undergoing a splicing reaction at
the protein level to produce both the 50 kDa spacer protein and the
69 kDa subunit of the vacuolar H+-ATPase. Shown are the residues at
splice junctions A and B, the protein sequence for the peptide that
spans the splice junction of the 69 kDa subunit (solid underlined) and
the amino-terminal protein sequence of the spliced spacer protein
(dashed underlined). The amino acid sequence of the peptide spanning
the spliced junction and the spacer protein amino terminus were
determined by protein sequencing. The splice junction cysteines are
numbered relative to the initiating methionine codon of TFPI.
Arrowheads indicate proposed cleavage points in Tfplp.
et al., 1990). In such a reaction, the 50 kDa spacer protein
is excised from the central portion of the precursor, while
the N- and C-domains are joined to create the vacuolar
H+-ATPase subunit (Figure
suggested that the joining of the N- and C-domains is via
the formation of a peptide bond (Kane et al., 1990). To test
this prediction, tryptic peptides from the native 69 kDa
vacuolar H+-ATPase subunit were separated by HPLC to
identify the peptide spanning the junction. Several peptides
that eluted near the position calculated for the junction
peptide were sequenced and all agreed with the predicted
amino acid sequence of regions of the N- and C-domains
of the 69 kDa polypeptide. The relevant peptide was
identified and is shown in Figure
are the amino acid sequences at the two splice junctions of
Tfplp. Edman degradation of the bond joining the N- and
C-domains demonstrates that the domains are linked via a
Only one cysteine residue was detected in the sequenced
junction peptide from the 69 kDa vacuolar H+-ATPase
subunit, yet a cysteine residue is present at each splice
junction in the Tfplp precursor (C284 and C738; Figure 1).
Mechanistically, it is important to determine which of the
two cysteine residues remains in the 69 kDa polypeptide as
it allows one to assign the peptide bonds that are broken in
the precursor and reformed in the spliced product. To
identify the position of the cysteine in the 50 kDa spacer
protein, this polypeptide was purified and subjected to amino-
terminal sequencing. Cysteine, and the subsequent sequence
shown in Figure
amino terminus of the spacer protein, thereby defining the
precise breakage points (arrowheads, Figure 1) and assigning
C738 to the spliced 69 kDa polypeptide.
1). Indirect evidence had
1 (solid underlined), as
1 (dashed underlined), was found at the
Removal of the N- and C-domains does not affect
To test the role of the Tfplp N- and C-domains in protein
were constructed in either or both
Fig. 2. Large deletions of the N- and C-domains do not inhibit protein
splicing. The schematic diagram shows the predicted protein encoded
by pAAC100: the 12 residues comprising the c-myc epitope, the distal
28 residues of the N-domain (N), the complete spacer domain
(SPACER), the proximal 13 residues of the C-domain (C) and 150
residues encoded by the LEU2 gene (LEU2). The strain SEY621 la-
tfpl\A was transformed with the following plasmids: lane 1, pRS316
(centromere containing vector with no insert; Sikorski and Heiter,
1989); lane 2, pPK26 (pRS316 containing TFP1): lane 3, pAAC100
(pRS316 containing the c-myc-N-spacer-C-Leu2p gene fusion). Cells
were grown to mid-log phase in liquid YEP media containing raffinose
and galactose (2% final concentration) for 6 h prior to harvesting. Cell
extracts were prepared as described, resolved by SDS-PAGE and
used in immunoblots probed with affinity-purified anti-spacer
domains. Large deletions in either the N- or C-domains did
not prevent protein splicing (data not shown). A chimeric
construct (pAAC100) was produced that combined the
separate deletions of the N- and C-domains. Given that the
N- and C-domains were now very small, the c-myc epitope
and a portion of yeast LEU2 gene were added to tag the
regions flanking the spacer by epitope or mass addition. In
addition to these unrelated sequences, the chimeric construct
encoded the complete Tfplp spacer flanked by the distal 28
residues of the N-domain and the proximal 13 residues of
the C-domain (Figure 2). The fusion protein was expressed
in a strain disrupted at the TFPJ locus QfpJA) and Western
blot analysis was performed on protein extracts with
antibodies directed against the spacer protein. If protein
splicing occurred at the predicted junctions, then the spacer
protein would be excised as a 50 kDa protein. If splicing
failed to occur, a 70 kDa protein is predicted that could be
detected with both anti-spacer protein and anti-c-myc an-
tibodies. No proteins were identified from the strain con-
taining the vector alone (Figure 2, lane 1), whereas the strain
expressing the fusion protein produced a 50 kDa protein,
which was detected with anti-spacer protein antibodies and
co-migrated with authentic spacer protein (Figure 2, lanes
2 and 3). The excision of the 50 kDa spacer protein from
such a construct predicts that a c-myc-tagged 20 kDa pro-
tein should result from the splicing reaction. However, the
anti-c-myc monoclonal antibody failed to detect any protein
resulting from expression of pAAC100 (data not shown),
suggesting that the expected c-myc-tagged 20 kDa protein
was unstable in yeast.
The spacer domain can splice from a new context
Truncations of the majority of both the N- and C-domains
did not affect splicing, and raised the possibility that the
A.A.Cooper et al.
Self-excision in protein splicing
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Received on January 19, 1993; revised on March 8, 1993