Profiles of Kex2p and K-V Transport
We developed previously an assay that measures cell-free transport of Kex2p from TGN donor membranes to PVC acceptor membranes containing the chimeric Kex2p substrate PSHA (see Materials and Methods for a description; Blanchette et al., 2004
). As an experimental refinement, we also reported that a Kex2p chimera (K-V) containing the cytosolic tail (C-tail) of Vps10p was also efficiently transported to the PVC (Abazeed et al., 2005
; A). Unlike Kex2p, Vps10p cycles directly between the TGN and the PVC in the absence of exchanges with the early endosome (Sipos et al., 2004
). Thus, transport of the K-V chimera in the cell-free assay should only occur via the direct pathway from the TGN to the PVC. The ability to measure transport of two distinct C-tails to the PVC permits an extended analysis of protein trafficking in the TGN-endosome system.
Figure 1. The C-tails of Kex2p and Vps10p regulate the rate of transport between the TGN and the PVC. (A) Schematic depiction of the domain composition of Kex2p, Vps10p, and the chimeric protein K-V. The numbers above each construct designate the N- and C-terminal (more ...)
To compare trafficking of Kex2p and K-V in vivo, we measured the relative rates of exit of these proteins from the pro-α-factor processing compartment(s) (TGN and/or early endosome) using the onset of impotence assay, a well-characterized plate-based mating assay (Redding et al., 1996
; Brickner and Fuller, 1997
; Sipos et al., 2004
). Patches of strain KRY24-2D (MAT
Δ), expressing wild-type Kex2p or K-V under GAL1
promoter control, were grown on galactose medium, shifted to glucose at various times to repress transcription, and then tested for their ability to mate with a MATa
tester strain (B). In this assay, as the concentration of Kex2p in the pro-α-factor processing compartment decreases below a level at which it becomes limiting for pro-α-factor cleavage, α-factor production, and thus mating efficiency, begins to decline as well (Wilcox et al., 1992
). The mating efficiency of cells in which K-V expression was shut off began to decline after 8 h on glucose medium, with residual mating occurring at the 10- and 12-h time intervals. In contrast, cells in which Kex2p expression was shut off continued to mate at later times, with effective mating occurring into the 12-h time point. In comparable assays, cells containing Kex2p lacking the C-tail lose mating competence within 2–4 h (Redding et al., 1996
). These results indicate that the Vps10 C-tail promotes localization of the Kex2p catalytic domain in the pro-α-factor processing compartment, but less efficiently than the Kex2p C-tail.
Next, we compared the function of the K-V chimera relative to wild-type Kex2p in cell-free transport. MSS containing Kex2 or K-V (C) was incubated with MSS containing PSHA, and PSHA cleavage was measured as a function of time at 30°C. Cell-free processing of PSHA by K-V was time-dependent, with processing achieving a plateau at ~20 min. Maximal cell-free processing by K-V was similar to that observed for Kex2p (~10%). However, differences in the kinetics of transport were observed. The Kex2p reaction was biphasic, consisting of an initial rapid phase followed by a secondary phase that occurred after a short lag (this is best observed in the 0- to 10-min time course in D). The K-V reaction exhibited a single phase, with PSHA processing occurring after an ~10-min lag. Thus, the Kex2p and Vps10p C-tails confer distinct kinetics of cell-free transport, in line with their distinguishable effects on pro-α-factor processing in vivo.
Gga2p Is Required for Cell-free TGN-to-PVC Transport of Kex2p and K-V
The yeast S. cerevisiae
contains two GGA
. Although deletion of either the GGA1
gene alone does not cause discernible sorting defects, cells lacking both GGA
genes have been shown to exhibit several post-Golgi trafficking defects (Black and Pelham, 2000
; Dell'Angelica et al., 2000
; Hirst et al., 2000
; Mullins and Bonifacino, 2001
). For example, gga1
Δ cells exhibit improper sorting of Vps10p, which binds both CPY and proteinase A as it cycles between the TGN and the PVC, and Kex2p, which also cycles between the TGN and endosomes and is required for the processing of pro-α-factor.
To study the role of the Gga proteins in Kex2p and K-V cell-free transport, a strain with a deletion of gga1
Δ and gga2
Δ was constructed. Previously, we found that deletion of several genes involved in vesicular transport resulted in elevated levels of Kex2p-independent cleavage of PSHA in vivo, preventing use of such strains in the cell-free reaction (our unpublished observation). To determine the level of in vivo PSHA cleavage in the gga1
Δ background, MSS isolated from strains JBY209 (GGA1 GGA2
) and MAY4 (gga1
Δ) expressing the PSHA fusion protein was subjected to IP by using anti-HA antibody in the presence of 1% (vol/vol) Triton X-100. As shown in A, deletion of gga1
Δ resulted in in vivo PSHA cleavage well above the level in JBY209. Kex2p-independent cleavage of PSHA in gga
null cells supports a role for the Gga proteins in Pep12p localization, consistent with previous reports that indicate a role for Gga proteins in the transport of Pep12p from the TGN to the PVC (Black and Pelham, 2000
Figure 2. Deletion of the GGAs results in increased PSHA processing and reduced cell-free transport of Kex2p and K-V. (A) PSHA is cleaved in vivo as a result of gga1 gga2 disruption. MSS samples prepared from JBY209 (kex2Δ GGA1+ GGA2+) and MAY8 (kex2Δ (more ...)
To test the role of the Gga proteins in cell-free transport, donor MSS from gga1Δ gga2Δ strains expressing either Kex2p or K-V and acceptor MSS from GGA1 GGA2 strains expressing PSHA were combined in transport reactions. Kex2p and K-V transport into the PVC was reduced by ~60% in these reactions (B). However, it was not clear whether the reduction in transport was due to the partial absence of Ggas in the cell-free reaction or to missorting of Kex2p and K-V in vivo.
To assess more directly whether Gga function was required during cell-free transport of Kex2p and K-V, we developed a strain with a disruption at the gga1
locus and an integrated 13 myc
peptide tag at the C terminus of Gga2p (strain MAY17). This approach permits analysis of what seems to be the dominant Gga protein Gga2p (Costaguta et al., 2001
). Gga2-13myc expression was confirmed by immunoblotting (A). The function of Gga2-13myc in vivo was tested using the CPY filter immunoblot assay (B). As expected, CPY was secreted by the gga1
Δ strain (Vps−
phenotype) (B). In contrast, MAY17 was Vps+
and did not exhibit elevated CPY secretion, indicating that Gga2-13myc is functional in vivo.
MSS from strains expressing Gga2p or Gga2-13myc was used for cell-free transport of Kex2p and K-V into the PVC in the absence and presence of anti-c-myc antibody (C). Anti-c-myc did not appreciably reduce transport in extracts prepared from Gga2p-expressing cells. However, in reactions containing MSS from Gga2-13myc expressing cells, Kex2p transport was reduced by ~50% and K-V was reduced by >80%. IP analysis demonstrated that anti-c-myc was able to recognize Gga2p-13myc during the course of the reaction (D). Because inhibition of Kex2p transport was incomplete, anti-c-myc was titrated into reactions containing Kex2p or K-V MSS, and dose–response curves were generated (E). Inhibition of Kex2p transport reactions plateaued at ~50%, suggesting that the residual transport represents a Gga2p-independent transport pathway for Kex2p. No plateau was observed in inhibition of K-V transport, suggesting a near complete dependence on Gga2p.
Dominant-Negative Gga2p VHS-GAT Inhibits Cell-free TGN-to-PVC Transport of Kex2p and K-V
Expression of a truncated form of human GGA1, consisting of the N-terminal VHS (VPS27
, and STAM
) and GAT (GGA
) domains, results in a dominant-negative phenotype, as measured by improper localization of the mannose-6-phosphate receptors (MPRs) (Puertollano et al., 2001
). Based on this, we tested a similar VHS-GAT form of yeast Gga2p for dominant-negative activity both in vivo and in the cell-free assay. Gga2p VHS-GAT is predicted to be recruited to membranes by ARF-GTP and phosphatidylinositol 4-phosphate (PI4P) and bind cargo through its VHS domain, but to lack the elements required for the recruitment of clathrin and accessory proteins involved in vesicle formation, the hinge and C-terminal γ-adaptin ear domain (Bonifacino, 2004
; Wang et al., 2007
; Demmel et al., 2008
To test the activity of Gga2p VHS-GAT in vivo, it was expressed under the control of constitutive and inducible promoters in wild-type cells. Expression from promoters with lower activity, the ADH
promoter in a high copy number (2μ) plasmid and the GAL1
promoter in a low copy number (CEN ARS
) plasmid, resulted in growth rates similar to wild-type cells. However, expression from 2μ vectors under the control of strong constitutive promoters (TEF
) or the strong inducible GAL1
promoter resulted in a slow growth phenotype (B). Given that deletion of two S. cerevisiae ARF
, is lethal (Stearns et al., 1990
), it is likely that the slow growth phenotype observed upon high level expression of Gga2p VHS-GATp is a result of sequestration of Arf.
High-level expression of Gga2p VHS-GAT was then tested for its effect on CPY sorting. As shown in C, expression of Gga2p VHS-GAT under ADH, TEF, or GPD promoter control led to secretion of CPY as measured by the colony immunoblot assay. Moreover, when Gga2p VHS-GAT was placed under the control of the inducible CUP1 promoter, growth on plates supplemented with 100 μM CuSO4 resulted in secretion of CPY (D). Cellular extracts from strains expressing Gga2-13myc under the GGA2 promoter and Gga2p VHS-GAT-6X His under ADH, TEF, and GPD promoters or Gga2p GST-VHS-GAT under the CUP1 promoter were resolved by SDS-PAGE, analyzed by immunoblotting using anti-c-myc and anti-His or anti-GST, and ratios of antibody signals were measured as described in Materials and Methods. Gga2-13myc to Gga2p VHS-GAT antibody signal ratios for ADH, TEF, GPD, and CUP1 were 1:5.2, 1:7.8, 1:6.4, and 1:9.4, respectively (D). Together, these results suggest that Gga2p VHS-GAT disrupts Vps10p transport between the TGN and the PVC in dominant-negative manner.
We then tested the ability of purified Gga2p VHS-GAT to inhibit cell-free transport of Kex2p and K-V into the PVC. Addition of bacterially expressed, purified Gga2p VHS-GAT (12 μg) to cell-free transport assays inhibited TGN-PVC transport reactions containing Kex2p donor membranes by ~60%, but it inhibited reactions containing K-V donor membranes >90% (A). Titration of Gga2p VHS-GAT protein showed that inhibition of Kex2p transport plateaued at ~55% of control (B). MSS containing Gga2-13myc expressed under the native GGA2 promoter and bacterially expressed, purified Gga2p VHS-GAT-6X His (12 μg) were resolved by SDS-PAGE, analyzed by immunoblotting using anti-c-myc and anti-His, and ratios of antibody signals were measured as described in Materials and Methods. Gga2-13myc to Gga2p VHS-GAT antibody signal ratios in cell-free transport assays were 1:13.7 (Kex2p) and 1:15 (K-V) (C). These results indicate that the relative amount of Gga2p VHS-GAT required to inhibit TGN-PVC transport in vitro is comparable with the relative amount required for dominant-negative function within the cell as measured by CPY secretion.
Together, inhibition of transport by anti-c-myc in reactions containing Gga2-13myc and by the Gga2p dominant-negative VHS-GAT in reactions containing wild-type Gga2p provides direct biochemical evidence for a role of Ggas in transport of Kex2p and Vps10p to the PVC. The full requirement for Gga in K-V transport (>90% inhibition by Gga2p VHS-GAT) indicates that this class of adaptors is required for direct TGN to PVC transport. In the case of Kex2p; however, partial inhibition suggests that in the cell-free assay, Kex2p is delivered to the PVC by distinct Gga-dependent and Gga-independent trafficking pathways.
Given that reactions with Kex2p donor membranes exhibited an initial rapid phase not seen with K-V membranes, we sought to determine whether the initial rapid phase of Kex2p transport corresponded to the Gga2p-independent pathway. When a time course of TGN-PVC transport using MSS containing Kex2p was performed in the presence of purified Gga2p VHS-GAT (12 μg), only the first, rapid phase of the reaction was observed (D). This result indicates that the early, rapid phase is Gga2p-independent. The second, slower phase resembles the time course with K-V donor membranes (D) and is Gga2p dependent.
AP-1 Is Not Required for Direct TGN-to-PVC Transport
Yeast AP-1 is heterotetrameric complex consisting of large (γ and β1), medium (μ1), and small (σ1) subunits. In yeast, sequence comparison with mammalian adaptins, genetic interactions with chc1ts
, and protein–protein interaction studies have established that Apl4p (γ), Apl2p (β1), Apm1p (μ1), and Aps1p (σ1) assemble to form AP-1 (Phan et al., 1994
; Rad et al., 1995
; Stepp et al., 1995
; Yeung et al., 1999
). It has also been shown that disruption of APL2
alone results in AP-1 mutant phenotypes with severity equivalent to that seen in cells that lack all four AP-1 subunits, suggesting a functional complex does not exist if APL2
is deleted (Yeung et al., 1999
). The requirement for APL2
in complex assembly allows for the assessment of AP-1 function in cell-free transport by manipulation of a single locus.
To determine the level of in vivo PSHA cleavage in the apl2Δ background, MSS isolated from strains JBY209 (APL2) and MAY8 (apl2Δ) expressing the PSHA fusion protein was subjected to IP by using anti-HA antibody in the presence of 1% (vol/vol) Triton X-100. As shown in A, deletion of APL2 did not result in increased cleavage of PSHA as had been observed in gga1Δ gga2Δ (A).
Figure 6. AP-1 is not required for cell-free TGN-to-PVC transport of Kex2p and K-V, but it is required for localization of Kex2p to the Gga-dependent donor compartment. (A) PSHA is not cleaved in vivo as a result of AP-1 disruption. MSS samples prepared from JBY209 (more ...)
To test the role of AP-1 in cell-free transport, MSS from apl2Δ strains expressing PSHA and either Kex2p or K-V were combined in transport reactions. In the apl2Δ reactions, Kex2p transport was diminished 60–80%, but K-V transport was unaffected compared with APL2+ controls (, B and G). These results indicate that cell-free transport of K-V from the TGN to the PVC is independent of AP-1 and suggest, further, that localization of K-V in donor membranes was not altered in vivo in the apl2Δ strain.
To determine whether residual PSHA cleavage seen with Kex2p-containing MSS from apl2
Δ cells represents transport into the PVC, we assayed for inhibition using F(ab) fragments of a monoclonal anti-Pep12p antibody. By phenotypic, structural, and sequence analysis, Pep12p is the likely heavy chain soluble N
-ethylmaleimide-sensitive factor attachment protein receptor of the PVC and mediates the fusion of incoming vesicular traffic into this compartment (Becherer et al., 1996
; Black and Pelham, 2000
; Gerrard et al., 2000
). C illustrates that the addition of anti-Pep12p F(ab) reduced transport by >95%. We conclude that Pep12p function is required for the cell-free delivery of Kex2p into PSHA-containing compartments in apl2
Δ MSS and that residual Kex2p transport in apl2
Δ MSS represents Kex2p transport into the PVC.
Although Kex2p transport into the PVC was reduced in apl2
Δ MSS, it was not clear whether reduction in transport was due to the absence of AP-1 function in the cell-free reaction or to missorting of Kex2p in vivo. The latter possibility would be consistent with studies demonstrating effects of AP-1 mutations both on Kex2-dependent processing in chc1ts
cells and on trafficking of other marker proteins between the TGN and early endosome (Rad et al., 1995
; Stepp et al., 1995
; Valdivia et al., 2002
; Foote and Nothwehr, 2006
). Further characterization of the AP-1–sensitive pathway in apl2
Δ MSS also permits an analysis of the role of AP-1 in anterograde traffic from the TGN/early endosome into the PVC; such a role for the AP-1 adaptor has remained elusive.
Given that reactions with Kex2p donor membranes exhibited an initial rapid phase not seen with K-V membranes (, C and D), we sought to determine whether the initial rapid phase of Kex2p transport corresponded to an apl2Δ-independent pathway. When a time course of TGN-PVC transport using MSS from apl2Δ strains expressing Kex2p and PSHA was performed, only the first, rapid phase of the reaction was observed (D). This result indicates that the early, rapid phase of Kex2p transport into the PVC is AP-1 independent.
As a direct test of a requirement for AP-1 function during the cell-free reaction, we determined the effect of adding an affinity-purified antibody against Apl2p. Even though this antibody recognized Apl2p when incubated with MSS under transport reaction conditions, addition of 14 μg of antibody had no effect on either Kex2p or K-V transport (, E and F).
To extend this analysis, the residual PSHA cleavage seen with Kex2p-containing MSS from apl2Δ cells was assayed for inhibition by Gga2p VHS-GAT. Addition of Gga2p VHS-GAT had no significant effect on the residual Kex2p transport in reactions with apl2Δ MSS (F). In contrast, addition of the Gga2p VHS-GAT inhibited transport reactions with K-V–containing MSS from apl2Δ cells to the same degree as seen with K-V–containing MSS from APL2+ cells (compare A and F). These results indicate that the effect of apl2Δ on transport of Kex2p in the cell-free assay is an indirect consequence of Kex2p mislocalization in vivo and that AP-1 plays no direct role in cell-free transport of Kex2p from the TGN to the PVC. Conversely, apl2Δ has no effect on either the localization of K-V in vivo or on cell-free transport of K-V.