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. 2025 Sep 3;28(10):113494.
doi: 10.1016/j.isci.2025.113494. eCollection 2025 Oct 17.

Identification of the client-binding site on the Golgi membrane protein adaptor Vps74/yGOLPH3

Agnieszka M Lesniak  1 , Ziyun Ye  1 , David K Banfield  1
Affiliations

Identification of the client-binding site on the Golgi membrane protein adaptor Vps74/yGOLPH3

Agnieszka M Lesniak et al. iScience. .

Abstract

Vps74 and its mammalian counterpart GOLPH3 are COPI associated protein sorting adaptors that function to maintain the cisternal distributions of a diversity of Golgi integral membrane protein clients by binding to their short cytoplasmically exposed N-termini. Here, we identify the client-binding site on yeast GOLPH3 (Vps74) which maps to two evolutionarily conserved loops on the membrane-facing surface, and includes residues mediating binding of GOLPH3s to PI4P, as well as the membrane-binding β hairpin. As an orthogonal approach, we isolated an inhibitory anti-Vps74 nanobody (which also binds to GOLPH3s) with which we corroborate the client-binding site and reveal that Sac1 and Arf1 binding to Vps74 blocks client access. We also identify an additional mode for the recruitment of Vps74 to Golgi membranes whereby the adaptor binds directly to its client N-termini. This study elucidates the molecular mechanism of Vps74 and identifies an inhibitory GOLPH3 nanobody with potential therapeutic applications.

Keywords: Biochemistry; cell biology; molecular biology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Vps74/mNeon fusion proteins are functional (A) Schematic representations of the various Vps74/mNeon fusion proteins used in this study. (B) VPS74-mNeon, mNeon-VPS74, and VPS74-mNeon-VPS74 can support the growth of the synthetic deletion mutant vps74Δ ted1Δ. 10-fold serial dilutions of the indicated yeast strains were spotted onto plates and thereafter incubated as indicated. n = 3. (C) vps74Δ ted1Δ cells expressing the various Vps74/mNeon fusion proteins are not temperature-sensitive for growth. 10-fold serial dilutions of the indicated yeast strains were spotted onto plates and thereafter incubated as indicated. n = 3. (D) Immunoblot and quantification of the steady-state levels of the various Vps74/mNeon fusion proteins in vps74Δ ted1Δ cells. Pgk1 serves as a gel load control. n = 3. (E) Immunoblots and quantification of steady-state levels of Dcr2 in vps74Δ cells expressing the various Vps74/mNeon fusion proteins. Pgk1 serves as a gel load control. n = 4. (F) Immunoblots and quantification of the steady-state levels of Mnn5 and Kre2 in cells expressing the various Vps74/mNeon fusion proteins. Pgk1 serves as a gel load control. n = 3. Data are presented as mean ± SD and each data point represents a biological replicate. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s multiple comparisons test (D) or Tukey’s multiple comparisons tests (E and F).
Figure 2
Figure 2
Vps74/mNeon fusion proteins do not differ in their Golgi cisternae distributions (A) Co-localization of the various Vps74/mNeon proteins in cells expressing mCherry-Sed5 (cis), Sec21-mScarlet (cis, medial and trans) or Sec7-mScarlet (trans) Golgi residents. Scale bars, 5 μm. n = 3. (B) Quantification of the number of mNeon puncta from experiments depicted in (A). (C) Quantification of the co-localization data for the various Vps74/mNeon proteins with cis, medial and trans Golgi markers from experiments depicted in (A). Data are presented as mean ± SD and each data point represents a biological replicate. Statistical analyses were performed using two-way ANOVA followed by Tukey’s multiple comparisons tests (B and C).
Figure 3
Figure 3
Over-expression of clients is sufficient to recruit Vps74 to Golgi membranes (A) Cells expressing GRX7, MNN5 and KRE2 from high copy number plasmids show more Golgi puncta. Scale bars, 5 μm. n = 9. (B) Over-expression of clients does not alter the steady-state levels of Vps74. Lefthand side immunoblot from one biological replicate. Righthand side quantification data from 4 biological replicates (n = 4). (C) GRX7 is a dosage suppressor of the temperature-sensitive growth phenotype of vps74-1 ted1Δ cells. 10-fold serial dilutions of the indicated yeast strains were spotted onto plates and thereafter incubated as indicated. n = 3. (D) The steady-state level of Grx7 is reduced in cells lacking VPS74. n = 3. Data are presented as mean ± SD and each data point represents a biological replicate. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s multiple comparisons tests (A and B) or two-tailed unpaired t test (D).
Figure 4
Figure 4
Overexpression of clients does not alter the cisternal distribution of Vps74 (A) Over-expression of GRX7, MNN5, and KRE2 is sufficient to recruit Vps74-mNeon to cis, medial, and trans Golgi cisternae. Scale bars, 5 μm. n = 3. (B) Client over-expression does not significantly alter the distribution of Vps74 across Golgi cisternae. Data are presented as mean ± SD and each data point represents a biological replicate. Statistical analyses were performed using two-way ANOVA followed by Sidak’s multiple comparisons test.
Figure 5
Figure 5
Amino acid substitutions to the PI4P-binding site and deletion of the β hairpin of Vps74 impact client-binding in vivo and in vitro (A) The R97 variant of Vps74 cannot support the growth of vps74Δ ted1Δ cells. 10-fold serial dilutions of the indicated yeast strains were spotted onto plates and thereafter incubated as indicated. n = 3. (B) Expression of the β hairpin deletion variant confers temperature-sensitivity for growth and calcofluor sensitivity in vps74Δ ted1Δ cells. 10-fold serial dilutions of the indicated yeast strains were spotted onto plates and thereafter incubated as indicated. n = 3. (C) Immunoblots and quantification of the steady-state levels of clients in vps74Δ cells expressing the Vps74 variants. Pgk1 serves as a gel load control. n = 6. (D) The R97, K178 R181, or β hairpin deletion (Δ197-208) variants show reduced binding to the N-terminus of Mnn4 in an in vitro mixing assay. Upper panel representative immunoblot, lower panel quantification from four biological replicates (n = 4). (E) Over-expression of Kre2 is not sufficient to recruit the R97, K178 R181, or β hairpin deletion (Δ197-208) variants to Golgi membranes. Scale bars, 5 μm. n = 3. (F) A heatmap depicting the impact of the Vps74 variants on client retention. Note that the steady-state levels of clients in cells lacking VPS74 (Empty vector, C) have been subtracted. Data are presented as mean ± SD and each data point represents a biological replicate. Statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple comparisons test (C) or Dunnett’s multiple comparisons test (D), or two-way ANOVA followed by Tukey’s multiple comparisons test (E).
Figure 6
Figure 6
PI4P binding may be a co-requisite for recruitment of Vps74 to Golgi membranes (A) Depiction of constructs used in B and C. (B) Inserting the PH domain of FAPP1 into Vps74-mNeon-Vps74 is sufficient to recruit Vps74 variants to the Golgi. Scale bars, 5 μm. n = 3. (C) The R97, K178 R181 and β hairpin variants of Vps74-mNeon-PH-Vps74 do not restore the steady-state levels of Mnn5 or Kre2. Pgk1 serves as a gel load control. n = 3. (D) A time course of Pik1 degron-mediated depletion reveals a role for PI4P and clients in the recruitment of Vps74 to Golgi membranes. Lefthand side depicts the experimental paradigm for degron-mediated degradation of Pik1. Righthand side summary of the time course data for 3 biological replicates: loss of Vps74 Golgi puncta, degradation of Pik1, and the steady-state levels of mNeon-Vps74. Data are presented as mean ± SD and each data point represents a biological replicate. Statistical analyses were performed using two-way ANOVA followed by Tukey’s multiple comparisons tests (B and C).
Figure 7
Figure 7
The client-binding site on Vps74 is located in two adjacent loops (A) The GOLPH3s share two conserved, surface exposed flexible loops located on the membrane-proximal surface. Note that Loop1 and Loop2 are adjacent to the PI4P binding site. (B) Docking client N-termini to Vps74 with AlphaFold2 identifies frequent contact sites in the client-binding adaptor interface. (C) A structural rendering of Vps74 in which the locations of Loop1 and Loop2 are shown relative to the Golgi membrane surface. Amino acids targeted for substitution are indicated along with depictions of their respective side chains. (D) Immunoblots and quantification of steady-state levels of clients in vps74Δ cells expressing the Loop1, Loop2, Loop1+Loop2 and W88 variants. Pgk1 serves as a gel load control. n = 6. (E) A heatmap summarizing the impact of the Vps74 Loop1 and Loop2 variants on client retention. Note that the steady-state levels of clients in cells lacking VPS74 (Empty vector, D) have been subtracted. (F) The impact of the Loop1 and Loop2 variants on Mnn4 binding to Vps74 are recapitulated in an in vitro mixing assay. n = 6. Data are presented as mean ± SD and each data point represents a biological replicate. Statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple comparisons tests (D and F).
Figure 8
Figure 8
The nanobody Vps74InNb#4 corroborates identification of the client-binding site on Vps74 (A) Expression of nanobody Vps74InNb#4 inhibits the growth of cells lacking TED1. 10-fold serial dilutions of the indicated yeast strains were spotted onto plates and thereafter incubated as indicated. n = 3. (B) Vps74InNb#4 binds to Vps74, human GOLPH3 and GOLPH3L. n = 3. (C) Expression of Vps74InNb#4 in wildtype cells reduces the steady-state levels of Mnn5, Kre2 and Dcr2. Pgk1 serves as a gel load control. n = 3. (D) Vps74InNb#4 reduces the binding of the N-terminus of Mnn4 to Vps74 in vitro. n = 3. (E) Expression of Vps74InNb#4 in wildtype cells blocks Golgi membrane recruitment of Vps74. Scale bars, 5 μm. n = 3. (F) Vps74InNb#4 binds to Loop1 and the β hairpin of Vps74. Vps74InNb#4 was docked with Vps74 using AlphaFold3. (G) Validation of the binding interface for Vps74InNb#4 on Vps74 with site-directed variants of Vps74. Data are presented as mean ± SD and each data point represents a biological replicate. Statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple comparisons test (C) or two-tailed unpaired t tests (C and D).
Figure 9
Figure 9
Client, Arf1, and Sac1 binding sites overlap on Vps74 (A) Sac1 competes with Mnn4 for binding to Vps74. n = 3. (B) Vps74InNb#4 does not block Sac1 binding to Vps74. n = 3. (C) Sac1 competes with Arf1-GTP for binding to Vps74. n = 3. (D) Vps74InNb#4 blocks binding of Arf1-GTP to Vps74. n = 5. (E) Arf1-GTP competes with Mnn4 for binding to Vps74. (F) The location of the client, Sac1 and Arf1-GTP binding sites on Vps74. Data are presented as mean ± SD and each data point represents a biological replicate. Statistical analyses were performed using two-tailed unpaired t tests (A, B, C, and D).
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