Phenotype of the ingls mouse
The spontaneous mutation ingls
(infantile gliosis) arose on the DBA/2J strain background and has been maintained by 23 generations of backcrossing to strain C57BL/6J to generate the congenic line B6. ingls
. Homozygous ingls
mutants exhibit reduced body size and diluted pigmentation that can be recognized as early as postnatal day 3 (P3) (). By P14, the mice exhibit a tremor and impaired motor function (Supplementary video
). The frequency of affected offspring born from crosses between heterozygous carriers is consistent with Mendelian expectation (29/140, P
=0.24). However, the maximal survival for ingls/ingls
homozygotes is 3 weeks, with continuous losses between P1 and P21. Histological analysis of the brain revealed greatly enlarged ventricles (, middle panel). Small areas with the appearance of spongiform degeneration are visible in several brain regions, including the thalamus, brain stem and cerebellar nucleus (Supplementary Figure S1A
). This pattern resembles the more widespread degeneration in the Fig4
-null mouse (Chow et al, 2007
). However, spinal motor neurons and sensory neurons in the DRG remain intact during the lifetime of the ingls
mutant (Supplementary Figure S1B
). Intense staining for glial fibrillary acidic protein (GFAP) is evident throughout the ingls
brain, suggesting widespread astrogliosis ( and Supplementary Figure S2A
). GFAP is sufficiently increased to be detected by western blot of cortical extracts (Supplementary Figure S2B
). Increased GFAP staining is evident as early as E14.5 (not shown).
Figure 1 ingls is caused by the missense mutation Vac14–L156R. (A) ingls homozygous mice at postnatal day 14 (P14) display reduced body weight and diluted coat colour. ingls/Vac14− compound heterozygotes at P14 are similar in appearance. (B) Severe (more ...)
Genetic mapping of ingls
mutation was first mapped to an 18 Mb interval on distal mouse chromosome 8, between microsatellite markers D8Mit86
, by genotyping 31 affected F2 offspring from a cross between DBA/2J.ingls
/+ and C57BL/6J (Bronson et al, 2003
). Genotyping additional markers on chromosome 8 reduced the nonrecombinant region to a 5.7 Mb interval with the following gene order: SNP1 (111.6 Mb)—1/62—ingls
—1/62—D8Mit88 (117.3 Mb) (Supplementary Figure S3A
). This nonrecombinant region contains 63 annotated genes (Mouse Build 37.2, www.ensembl.org
Identification of a mutation in Vac14
gene spans the region between 113.1 and 113.2 Mb on chromosome 8, within the ingls
nonrecombinant region. Based on similarities between the ingls
phenotype and null mutations of Vac14
(Chow et al, 2007
; Zhang et al, 2007
), we tested Vac14
as a candidate gene for ingls
The 19 exons of Vac14
were amplified from genomic DNA of an affected ingls
homozygote and sequenced. Comparison with the wild-type sequence from strain C57BL/6J (Build 37.2, www.genome.ucsc.edu
) identified the nucleotide substitution c.467T>G in exon 4 (). Strain DBA/2J, the strain of origin of the ingls
mutation, contains the wildtype residue (not shown). The T>G substitution destroys a Sac
I endonuclease site, which was used to genotype the Vac14ingls
allele. The mutation results in substitution of an arginine residue for the evolutionarily invariant residue leucine 156 (). Western blot analysis of brain extracts revealed normal abundance of Vac14 protein in the mutant brain ().
Noncomplementation of ingls and a Vac14-null allele
To confirm the causal function of the Vac14-L156R mutation in the ingls
phenotype, we assessed the ability of the Vac14ingls
allele to complement the recessive lethality of the null allele Vac14β−geo
) (Zhang et al, 2007
). Heterozygous ingls
) were crossed with null heterozygotes (Vac14+/−
). The Vac14ingls/−
compound heterozygotes were born in the predicted Mendelian ratio of 25% but did not survive to weaning, showing that ingls
is allelic with the null allele (Supplementary Figure S3B
). The compound heterozygotes reproduced the phenotype of the ingls
homozygote, with reduced body size and diluted pigmentation (), as well as enlarged ventricles (). The phenotypic similarity between Vac14ingls/−
compound heterozygotes and ingls
homozygotes, the failure of the ingls
allele to rescue the null homozygote, and the mapping of ingls
to an interval of chromosome 8 that includes Vac14
, support the identification of Vac14-L156R as the ingls
mutation. The survival of the compound heterozygotes to P14, compared with the neonatal lethality of null homozygotes, shows that the Vac14L156R
allele retains partial function.
Reduced levels of PI(3,5)P2 and vacuolization of cultured ingls fibroblasts
Levels of phosphoinositide lipids were assessed in cultured ingls fibroblasts. In wild-type fibroblasts, PI(3,5)P2 comprised 0.04% of total PI. In mutant ingls fibroblasts, PI(3,5)P2 was reduced to 50% of normal, whereas the levels of PI3P, PI4P and PI(4,5)P2 were unaffected ().
There is accumulation of intracellular cytoplasmic vacuoles in the cultured fibroblasts from ingls
mice (, bottom). The vacuolated appearance of the ingls
fibroblasts is similar to the fibroblasts from Vac14
- and Fig4
-null mice, indicating that the reduced level of PI(3,5)P2
is responsible for the similarity (Chow et al, 2007
; Zhang et al, 2007
). Transfection of a construct expressing a wild-type human Vac14–mCit fusion protein reduced the frequency of vacuolized ingls
fibroblasts from 20 to 5% (). Correction by wild-type Vac14 shows that the Vac14–L156R mutation is responsible for the ingls
cellular phenotype. The stability of the mutant protein in vivo
indicates that the Vac14–L156R protein is functionally impaired.
Prediction that Vac14 is composed entirely of HEAT repeats places the L156R mutation in repeat 4
HEAT repeats contain two anti-parallel helices connected by a short loop. These units often appear as a tandem series and provide surfaces for protein–protein interactions (Groves et al, 1999
; Andrade et al, 2000
). Previous analysis of the Vac14p sequence identified 1–3 HEAT repeats (Dove et al, 2002
). As HEAT repeat sequences are degenerated, many bona fide repeats are not detected by motif detection programs.
Analysis of the Vac14 protein sequence with the repeat finding program REP set at a confidence threshold of Pθ
) (Andrade et al, 2000
) predicted eight HEAT repeats in the Saccharomyces cerevisiae
Vac14p sequence. One additional repeat was identified by REP in Drosophila melanogaster
and Schizosaccharomyces pombe
sequences and, by sequence similarity, was found in the S. cerevisiae
sequence. A second analysis incorporated Vac14p sequences from 17 diverse species. Predicted HEAT repeats were defined by three criteria: (1) identification by REP in one or more species using no confidence threshold; (2) secondary structure prediction of two helices connected by a short loop; (3) alignment by visual inspection with HEAT repeat consensus sequences from the Pfam database. This analysis identified 21 potential HEAT repeats in S. cerevisiae
Vac14p (). Many exhibit sequence similarity in all 17 species (not shown). Yeast Vac14p is thus most likely to contain a minimum of 9 HEAT repeats and as many as 21 repeats, which would comprise the entire protein. Analysis of human Vac14 predicted 17 repeats, 15 of which have homologues in S. cerevisiae
Vac14p. The L156R mutation is located in helix B of HEAT repeat 4 (). A similar analysis accurately predicted the 50 HEAT repeats of TOR1 (Perry and Kleckner, 2003
Figure 2 Vac14 nucleates the Fab1 complex. (A) Vac14 contains 21 and 17 HEAT repeats in S. cerevisiae (top) and mammalian Vac14 (bottom), respectively. Identical colours indicate homologous repeats between S. cerevisiae and mammalian Vac14. Red asterisk and blue (more ...)
The L156R mutation disrupts Vac14 interaction with Fab1 but not Fig4
HEAT repeat proteins usually function as scaffolds with multiple binding partners (Groves et al, 1999
; Andrade et al, 2000
). The prediction that Vac14 may be composed entirely of HEAT repeats suggested that Vac14 may bind proteins that regulate PI(3,5)P2
levels. Previous sedimentation and coimmunoprecipitation analysis demonstrated that mammalian Vac14, Fab1, and Fig4 associate with each other (Sbrissa et al, 2007
). We tested potential interactions using the yeast two-hybrid test and found that human Vac14 interacts with Fab1 and Fig4 (). The ingls
mutation, Vac14-L156R, specifically disrupts the interaction between Vac14 and Fab1 but retains interaction with Fig4 (). A third interaction of yeast Vac14p is self-association (Dove et al, 2002
). We find that human Vac14 also interacts with itself and this interaction is unaffected by the Vac14–L156R mutation (). These data suggest that disruption of the interaction between Fab1 and Vac14 is the molecular basis for the ingls
Vac14p interacts with the known protein regulators of PI(3,5)P2
To determine whether Vac14p interacts with the other proteins known to regulate PI(3,5)P2 levels, we tested Vac7p and Atg18p, two additional regulators of yeast Fab1p. As Fab1p and Vac7p are large proteins, we performed a yeast two-hybrid test with constructs that expressed overlapping peptides from conserved regions of Fab1p and Vac7p. We found that Fab1p peptide 538–1085 and Vac7p peptide 394–918 interacted with yeast Vac14p in the yeast two-hybrid test (). Vac14p also interacts with full-length constructs of Fig4p and Atg18p and with itself ().
To determine whether each protein colocalizes with Vac14p in vivo
, we generated a chromosomal integrant of a Vac14p fusion construct containing the fluorescent tag RFP–mCherry. Consistent with the yeast two-hybrid studies, Vac14p–mCherry colocalized on the vacuole membrane with GFP- or Venus-tagged Fab1p, Fig4p, Vac7p, and Atg18p (Supplementary Figure S5
Fab1 and its regulators reside within a large protein complex
Coimmunoprecitation experiments were performed to test the model that Vac14p nucleates a large complex (). Using the tagged proteins Vac14p–HA and Vac14p–V5, we found that immunoprecipitation with anti-HA antibody coprecipitated Vac14p–V5 (Supplementary Figure S4A
). Furthermore, Fab1p containing a C-terminal tandem affinity purification (TAP) tag (Fab1–TAP) coprecipitated Vac14p–V5 (Supplementary Figure S4B
). Moreover, immunoprecipitation of Vac14p coprecipitates Fig4p (Supplementary Figure S4C
). In addition, precipitation of Fig4p coprecipitates Fab1p (Supplementary Figure S4D
). These interactions were reported independently (Rudge et al, 2004
; Botelho et al, 2008
), and it was proposed that Fab1p, Vac14p, and Fig4p form a ternary complex (Botelho et al, 2008
Immunoprecipitation of Vac14p–Venus also coprecipitates Vac7p (). These coprecipitation experiments demonstrate that Vac14p interacts with itself, Fab1p, Fig4p, and Vac7p. We did not observe coprecipitation of Atg18p with Vac14p, Fab1p-TAP, or Vac7p (not shown).
To determine whether the proteins form a large complex, we used sedimentation analysis of detergent solubilized yeast cell lysates. When lysates were fractionated on a 10–50% glycerol gradient, Fab1 was distributed in two peaks (). The smaller peak, fraction 3, contained monomeric Fab1p (predicted molecular weight 257 kDa). The larger peak, fractions 6–8, contained Fab1p in a complex that sediments more slowly than the 670 kDa molecular weight standard in fractions 4 and 5. Although the predicted molecular weights of Vac7p, Fig4p, and Vac14p are 128, 101, and 99 kDa, respectively, significant proportions of these proteins comigrated with the large Fab1p complex: Vac7p, in fractions 6 and 7; Fig4p, in fractions 6–8; and Vac14p, in fractions 5–8. That the peak fractions for Fab1p, Vac7p, Fig4p, and Vac14p overlap but are not identical may be explained by the presence of a mixture of partial and complete complexes. For example, the ternary complex of Fab1p, Vac14p, and Fig4p is found exclusively on the membrane of the vacuole, whereas Vac14p and Fig4p associate with each other in the cytosol (Botelho et al, 2008
Most of the Atg18p (55 kDa) behaved as a monomer (fractions 1 and 2), but some Atg18p comigrated with the complex (fractions 5–7), suggesting that this small fraction is also part of the complex. Alternatively, the Atg18 in fraction 6 could be trailing from the earlier fractions. However, note that the intensity of fraction 6 is the same as fraction 5. Genetic evidence (below) strongly suggests that a portion of Atg18p is part of the Vac14p complex. Atg18p has a second distinct function in autophagy and the large proportion of monomeric protein may be related to that function.
The above-mentioned results are consistent with the model that Vac14p nucleates a large complex containing Fab1p, Fig4p, Vac7p, Vac14p, and possibly Atg18p. Rather than pursuing analysis of the complex through further biochemical investigations, we identified and characterized mutations that partially disrupt the complex as a means to determine the functional significance of complete and partial complexes, and also map the interacting domains.
Fab1p, Vac14p, and Fig4p are required to form the core PI(3,5)P2 regulatory complex
Fab1p does not interact with Fig4p in a yeast two-hybrid test. However, Fab1p–TAP coprecipitates with Fig4p, suggesting that the two proteins could interact indirectly. Indeed, coprecipitation of Fig4p by Fab1p–TAP required the presence of Vac14p and did not occur in the vac14Δ
strain (Supplementary Figure S4E
). Unexpectedly, coprecipitation of Fab1p–TAP with Vac14p required the presence of Fig4p (Supplementary Figure S4F
). These data indicate that Fab1p requires both Vac14p and Fig4p to form a ternary complex in vivo
. Localization experiments indicate that formation of the ternary complex is required for normal localization of members of the complex to the vacuole membrane (). Note that in each mutant, vac14Δ
, and fab1Δ
, there is mislocalization to the cytoplasm of significant amounts of the remaining members of the complex.
Figure 3 The localization of Fab1p, Fig4p, and Vac14p to the vacuole membrane requires the presence of all three proteins. Cells labelled with FM4-64 (red) to visualize the vacuole membrane. (A) Wild-type strains with FAB1–3xGFP, FIG4–3xGFP, or (more ...)
Vac7p binds Vac14p in yeast two-hybrid and coimmunoprecipitation experiments ( and 2E). To determine whether Vac7p is required for formation of the Fab1p/Vac14p/Fig4p complex, we carried out coprecipitation experiments in vac7Δ
cells. Interaction of Fab1p with Vac14p (Supplementary Figure S6A
) and Fig4p (Supplementary Figure S6B
) did not require Vac7p. Localization of Fig4p, Vac14p, and Fab1p to the vacuolar membrane was independent of Vac7p (Supplementary Figure S6C
). Similarly, Vac7p localization to the vacuole membrane is independent of Vac14p and Fig4p (Supplementary Figure S6C
). Although two-hybrid studies support the proposal that Atg18p is part of the complex, this was not observed by coprecipitation. To further investigate the association of Atg18p with the complex, we examined the effects of Vac14p mutations (below).
The Vac14–ingls mutation disrupts interaction with yeast Fab1p, Vac7p, and Atg18p
To further examine the molecular consequences of the ingls Vac14–L156R mutation, we generated the equivalent yeast mutant, vac14–L149R. The L149R mutation is located in HEAT repeat 4 () and does not reduce protein stability (). The vac14Δ mutant has grossly enlarged vacuoles compared with the smaller and multilobed wild-type vacuoles (). When vac14–L149R is present as the sole copy of VAC14, vacuole size and morphology are intermediate between the multilobed wild-type and the vac14Δ mutant, consistent with partial loss of function ().
Figure 4 The vac14–L149R mutant is defective in its association with Fab1p. (A) Schematic of the vac14–L149R mutant. Red asterisk: mutation site. (B) vac14–L149R protein is stable. Cell lysates from a vac14Δ strain expressing pRS413– (more ...)
In a yeast two-hybrid test, vac14p–L149R was defective in binding Fab1p, Vac7p, and Atg18p, although retaining normal interaction with Fig4p and with itself (). vac14p–L149R also failed to coprecipitate with Fab1p–TAP (). In the presence of vac14p–L149R, Fab1p did not localize to the vacuole membrane (). Thus, HEAT repeat 4 is critical for the interaction between Vac14p and Fab1p and is required for the intracellular localization of Fab1p.
In contrast, vac14p–L149R did coprecipitate with Fig4p and with itself (Supplementary Figure S7
), indicating that the Fig4p and Vac14p interaction domains are located in a different region of Vac14p from the L149 mutation.
To examine the effect of loss of Fab1p from the PI(3,5)P2
regulatory complex, we examined the in vivo
levels of PI(3,5)P2
. Under basal conditions, PI(3,5)P2
comprises approximately 0.04% of total PI lipid in the vac14Δ
strain expressing wild-type VAC14
from a plasmid. When cells are exposed to hyperosmotic stress, PI(3,5)P2
increases 10-fold to approximately 0.5% of total PI lipid within 10 min, then decreases to 0.3% at 20 min, and to 0.09% at 30 min (, Supplementary Table S1
). (The 10-fold increase in this wild-type strain compared with a more typical 20-fold increase may be a consequence of the expression of VAC14
from a plasmid rather than from its chromosomal location.) In the vac14Δ
strain expressing vac14–L149R
comprises 0.03% of the PI lipid under basal conditions and increases only two-fold to 0.06% after 10 min of hyperosmotic stress. Thus, dissociation of Fab1p from the complex through loss of interaction between Vac14p and Fab1p results in defective activation of Fab1 lipid kinase activity in response to hyperosmotic shock. That the modest increase in PI(3,5)P2
is sustained for at least 30 min suggests that regulation of Fig4p lipid phosphatase activity may also be impaired by the loss of association of Fab1p and Vac14p.
The fab1-2 mutant reveals that activation of Fab1p requires its association with the Vac14p complex
We sought to identify a mutation in Fab1p that disrupted its interaction with Vac14p, and found that the fab1-2
mutant (Yamamoto et al, 1995
) is specifically defective in this interaction. We sequenced the mutant fab1-2
, and identified a single missense mutation, G864E, in the CCT domain (). The mutant protein is stable in vivo
() and retains normal kinase activity in vitro
(Supplementary Figure S8
). The CCT domain of Fab1p shares homology with the GroEL and the CCT family of Hsp60 chaperones, suggesting that it could be involved in protein–protein interactions. The G864E mutation is located within Fab1p peptide 538–1085, which interacts with Vac14p in a yeast two-hybrid test ( and ). We therefore tested the effect of this mutation on the interaction between Fab1 and Vac14. In the yeast two-hybrid test, fab1–G864E
does not interact with VAC14
(). Wild-type Fab1p coimmunoprecipitates with Vac14p–Venus, but the Fab1p–G864E mutant does not (). Thus, the G864E mutation disrupts interaction of Fab1p with Vac14p.
Figure 5 Formation of a ternary complex of Fab1p, Vac14p, and Fig4p is required for Fab1p kinase activity in vivo. (A) Schematic of the fab1-2 allele; purple asterisk, the G864E mutation. (B) Fab1-2 protein is present at wild-type levels. Cell lysates analysed (more ...)
Fab1p coimmunoprecipitates with Fig4p–3XGFP (). However, the Fab1p–G864E mutant protein does not coprecipitate with Fig4p–3XGFP (). This further supports the model that interaction of Fab1p with Fig4p is dependent on interaction of Fab1p and Fig4p with Vac14p (Supplementary Figure S4E and F
). These coprecipitation results are similar to those observed for fab1p–T1017I, another point mutation in the CCT domain (Botelho et al, 2008
The fab1-2 mutant protein fails to localize to the vacuole membrane (). Localizations of Vac14p–Venus and Fig4p–3xGFP to the vacuole membrane are also defective in the presence of fab1-2 protein (). Formation of the ternary complex thus appears to be required for correct localization of each component.
To determine the importance of formation of the ternary complex for the hyperosmotic stress-induced activation of Fab1p, we tested the effect of osmotic stress on PI(3,5)P2
levels in the fab1-2
mutant. In the wild-type strain, the basal levels of PI(3,5)P2
, 0.05% of total PI lipid, increase 20-fold to 1.15% within 10 min after hyperosmotic stress, then decrease to 0.32% at 20 min and 0.11% at 30 min (, Supplementary Table S1
). In the fab1-2
mutant, the basal levels of PI(3,5)P2
, 0.033%, increased only two-fold after 10 min, similar to the vac14–L149
mutant described above. These data support the conclusion that association with the complex is required for the hyperosmotic stress-induced activation of Fab1p and for the physiological regulation of PI(3,5)P2
The vac14-2 mutant disrupts interaction with Vac7p and Atg18p and results in defective activation and inhibition of Fab1p
Vac7p is a major activator of yeast Fab1p, and basal levels of PI(3,5)P2
are significantly reduced in a vac7Δ
mutant, resulting in enlarged vacuoles (Bonangelino et al, 1997
; Duex et al, 2006b
). We screened for gain-of-function mutations of Vac14p that could rescue the low levels of PI(3,5)P2
and reduce the enlarged vacuoles in vac7Δ
cells. Random PCR mutagenesis of VAC14
was performed and the mutants were transformed into a vac14Δ, vac7Δ
strain. Mutants that restored growth at 37°C and partially corrected the enlarged vacuoles were isolated. When the strongest of these mutants, vac14-2
, was expressed in a vac14Δ
strain, vacuole size was smaller than in wild-type cells (), suggesting that PI(3,5)P2
level is increased in this mutant. Direct measurement demonstrated a two-fold increase of the basal level of PI(3,5)P2
in the vac14-2
mutant (, Supplementary Table S1
). However, when the vac14-2
mutant was treated with hyperosmotic stress, the level of PI(3,5)P2
increased only 1.7-fold, compared with an 8-fold increase in the wild-type strain (). There was no change in the steady-state level of vac14-2 protein compared with wild type (). The abnormal regulation of PI(3,5)P2
thus appears to be caused by altered function of the vac14-2 protein.
Figure 6 Vac7p and Atg18p function through Vac14p. (A) vac14-2 has smaller than normal vacuoles, consistent with elevated basal levels of PI(3,5)P2. vac14Δ cells expressing pRS416–VAC14 or pRS416–vac14-2 labelled with FM4-64. (B) vac14-2 (more ...)
In a yeast two-hybrid test, vac14 -2 is specifically defective in interaction with Vac7p and Atg18p (). Interaction of vac14-2 protein with Vac14p, Fig4p, and Fab1p is normal and formation of the ternary complex is unaffected. The vac14-2 protein also coprecipitates Fab1p to the same extent as wild-type Vac14p ().
vac14-2 contains four mutations: H56Y, R61K, Q101R, and L329I (). Three of these mutations, H56, R61, and Q101, are located in HEAT repeat loops 2 and 3. These residues exhibit evolutionary conservation. Tested singly in a yeast two-hybrid test, each mutation perturbed the interaction of Vac14p with Vac7p and Atg18p, without disrupting interaction with its other binding partners (not shown). Thus all three residues are important for Vac14p interaction with Vac7p and Atg18p. L329 alone had no effect on the interaction of Vac14p with its known binding partners (data not shown). These results indicate that HEAT repeats 2 and 3 directly contact Vac7p and Atg18p, but do not contact Fab1.
The elevation in basal levels of PI(3,5)P2 in the vac14-2 mutant is consistent with the model that negative regulation of Fab1p by Atg18p requires binding of Atg18p to Vac14p. Whereas the yeast two-hybrid test detected interaction of Atg18p with Vac14p, this was not observed in pull-down experiments. Thus, it is informative that a mutation in Vac14p that interrupts its ability to interact with Atg18p also results in PI(3,5)P2 levels that are consistent with a partial release of Atg18p inhibition of Fab1p. The vac14-2 mutant provides additional support for the hypothesis that Atg18p interacts with Vac14p.
The vac14-2 defect in osmotic stress-induced elevation of PI(3,5)P2 supports the model that Vac7p, a positive regulator of Fab1p, exert an effect through binding to Vac14p. The association of Vac7p with wild-type Vac14p was demonstrated in the yeast two-hybrid test and by coimmunoprecipitation ().
The Fig4p-binding site on Vac14p is distinct from the Fab1p-, Vac7p-, and Atg18p-binding sites
Fab1p and Vac7p bind near the N terminus of Vac14p. On the basis of the predicted HEAT motifs of Vac14p, we divided the protein into two overlapping constructs (). HEAT repeats 1–10 interact with Fab1p and Vac7p but not with Fig4p. HEAT repeats 10–21 interact only with Fig4p. These results demonstrate that Fab1p and Vac7p bind to the N-terminal half of Vac14p, whereas Fig4p binds to a distinct site in the C-terminal half. Atg18p binds full-length Vac14p but does not bind to either half of the molecule. On the basis of the vac14-2 mutant, we predict that contact points for Atg18p include the intrarepeat loops of HEAT repeats 2 and 3.
Figure 7 Yeast two-hybrid analysis of the minimal region of Vac14p required for interaction with Fab1p, Fig4p, Vac7p, and Atg18p. Lines above the schematic indicate the minimal region of Vac14p required for binding with each protein. Full-length VAC14 or truncated (more ...)