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Ciliary membrane proteins traffic through the Golgi via a Rabep1/GGA1/Arl3-dependent mechanism.

Kim H, Xu H, Yao Q, Li W, Huang Q, Outeda P, Cebotaru V, Chiaravalli M, Boletta A, Piontek K, Germino GG, Weinman EJ, Watnick T, Qian F - Nat Commun (2014)

Bottom Line: PC1 must also be proteolytically cleaved at a GPS site for this to occur.Using yeast two-hybrid screening coupled with a candidate approach, we identify a Rabep1/GGA1/Arl3-dependent ciliary targeting mechanism, whereby Rabep1 couples the polycystin complex to a GGA1/Arl3-based ciliary trafficking module at the TGN.This study provides novel insights into the ciliary trafficking mechanism of membrane proteins.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

ABSTRACT
Primary cilia contain specific receptors and channel proteins that sense the extracellular milieu. Defective ciliary function causes ciliopathies such as autosomal dominant polycystic kidney disease (ADPKD). However, little is known about how large ciliary transmembrane proteins traffic to the cilia. Polycystin-1 (PC1) and -2 (PC2), the two ADPKD gene products, are large transmembrane proteins that co-localize to cilia where they act to control proper tubular diameter. Here we describe that PC1 and PC2 must interact and form a complex to reach the trans-Golgi network (TGN) for subsequent ciliary targeting. PC1 must also be proteolytically cleaved at a GPS site for this to occur. Using yeast two-hybrid screening coupled with a candidate approach, we identify a Rabep1/GGA1/Arl3-dependent ciliary targeting mechanism, whereby Rabep1 couples the polycystin complex to a GGA1/Arl3-based ciliary trafficking module at the TGN. This study provides novel insights into the ciliary trafficking mechanism of membrane proteins.

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Interaction of PC1 cytoplasmic C-terminal tail with Rabep1.(a) Yeast two-hybrid screening of a rat brain cDNA library using human PC1 215-amino-acid C-terminal tail as bait (PC1-CT) identified a clone (TH10-3) corresponding to amino acids 465 to 799 of Rabep1 with extensive coiled-coils69. (b) Mapping of Rabep1 interaction region by yeast two-hybrid assay using a series of PC1-CT deletion constructs22. The coiled-coil domain is indicated. PC1-M7 contains proline at a and d positions of the heptad, disrupting α-helical structure of the coiled-coil domain. PC1-P89 contains a germline mutation R4227X2227. PC1-C15 contains a somatic mutation found in a renal cyst resulting in reading-frame shift2270. Positive interaction was scored by both growth on triple selective medium and positive β-gal activity. PC2 C-terminal tail (PC2-CT) or Fos served as negative controls. Interactions of the PC1 constructs with PC2-CT are shown as a comparison. (c) Co-immunoprecipitation of endogenous PC1 with Rabep1, but not with Arf4 in CD cells.
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f6: Interaction of PC1 cytoplasmic C-terminal tail with Rabep1.(a) Yeast two-hybrid screening of a rat brain cDNA library using human PC1 215-amino-acid C-terminal tail as bait (PC1-CT) identified a clone (TH10-3) corresponding to amino acids 465 to 799 of Rabep1 with extensive coiled-coils69. (b) Mapping of Rabep1 interaction region by yeast two-hybrid assay using a series of PC1-CT deletion constructs22. The coiled-coil domain is indicated. PC1-M7 contains proline at a and d positions of the heptad, disrupting α-helical structure of the coiled-coil domain. PC1-P89 contains a germline mutation R4227X2227. PC1-C15 contains a somatic mutation found in a renal cyst resulting in reading-frame shift2270. Positive interaction was scored by both growth on triple selective medium and positive β-gal activity. PC2 C-terminal tail (PC2-CT) or Fos served as negative controls. Interactions of the PC1 constructs with PC2-CT are shown as a comparison. (c) Co-immunoprecipitation of endogenous PC1 with Rabep1, but not with Arf4 in CD cells.

Mentions: To identify the molecules that mediate polycystin complex ciliary trafficking, we performed a yeast two-hybrid screen for PC1-interacting proteins using the entire 215-amino-acid C-terminal tail as bait. Thirteen unique cDNA clones were identified that specifically interact with the PC1 C-terminus. One of these clones corresponded to amino acids 465 to 799 of Rabep1 (Fig. 6a), an effector of multiple Rab GTPases involved in various steps of intracellular vesicular trafficking40. This protein was chosen for further analysis. We used mutational analysis to map the Rabep1 interaction region (Fig. 6b) and showed that the C-terminal 89 amino acids containing the intact coiled-coil domain of PC1 (PC1-M1/M2) was sufficient for interaction with Rabep1. The binding sites for Rabep1 and PC2 overlap but are not identical, as the C-terminal 41 amino acids of PC1 (PC1-M3) was required for Rabep1 interaction, but not for PC2.


Ciliary membrane proteins traffic through the Golgi via a Rabep1/GGA1/Arl3-dependent mechanism.

Kim H, Xu H, Yao Q, Li W, Huang Q, Outeda P, Cebotaru V, Chiaravalli M, Boletta A, Piontek K, Germino GG, Weinman EJ, Watnick T, Qian F - Nat Commun (2014)

Interaction of PC1 cytoplasmic C-terminal tail with Rabep1.(a) Yeast two-hybrid screening of a rat brain cDNA library using human PC1 215-amino-acid C-terminal tail as bait (PC1-CT) identified a clone (TH10-3) corresponding to amino acids 465 to 799 of Rabep1 with extensive coiled-coils69. (b) Mapping of Rabep1 interaction region by yeast two-hybrid assay using a series of PC1-CT deletion constructs22. The coiled-coil domain is indicated. PC1-M7 contains proline at a and d positions of the heptad, disrupting α-helical structure of the coiled-coil domain. PC1-P89 contains a germline mutation R4227X2227. PC1-C15 contains a somatic mutation found in a renal cyst resulting in reading-frame shift2270. Positive interaction was scored by both growth on triple selective medium and positive β-gal activity. PC2 C-terminal tail (PC2-CT) or Fos served as negative controls. Interactions of the PC1 constructs with PC2-CT are shown as a comparison. (c) Co-immunoprecipitation of endogenous PC1 with Rabep1, but not with Arf4 in CD cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4237283&req=5

f6: Interaction of PC1 cytoplasmic C-terminal tail with Rabep1.(a) Yeast two-hybrid screening of a rat brain cDNA library using human PC1 215-amino-acid C-terminal tail as bait (PC1-CT) identified a clone (TH10-3) corresponding to amino acids 465 to 799 of Rabep1 with extensive coiled-coils69. (b) Mapping of Rabep1 interaction region by yeast two-hybrid assay using a series of PC1-CT deletion constructs22. The coiled-coil domain is indicated. PC1-M7 contains proline at a and d positions of the heptad, disrupting α-helical structure of the coiled-coil domain. PC1-P89 contains a germline mutation R4227X2227. PC1-C15 contains a somatic mutation found in a renal cyst resulting in reading-frame shift2270. Positive interaction was scored by both growth on triple selective medium and positive β-gal activity. PC2 C-terminal tail (PC2-CT) or Fos served as negative controls. Interactions of the PC1 constructs with PC2-CT are shown as a comparison. (c) Co-immunoprecipitation of endogenous PC1 with Rabep1, but not with Arf4 in CD cells.
Mentions: To identify the molecules that mediate polycystin complex ciliary trafficking, we performed a yeast two-hybrid screen for PC1-interacting proteins using the entire 215-amino-acid C-terminal tail as bait. Thirteen unique cDNA clones were identified that specifically interact with the PC1 C-terminus. One of these clones corresponded to amino acids 465 to 799 of Rabep1 (Fig. 6a), an effector of multiple Rab GTPases involved in various steps of intracellular vesicular trafficking40. This protein was chosen for further analysis. We used mutational analysis to map the Rabep1 interaction region (Fig. 6b) and showed that the C-terminal 89 amino acids containing the intact coiled-coil domain of PC1 (PC1-M1/M2) was sufficient for interaction with Rabep1. The binding sites for Rabep1 and PC2 overlap but are not identical, as the C-terminal 41 amino acids of PC1 (PC1-M3) was required for Rabep1 interaction, but not for PC2.

Bottom Line: PC1 must also be proteolytically cleaved at a GPS site for this to occur.Using yeast two-hybrid screening coupled with a candidate approach, we identify a Rabep1/GGA1/Arl3-dependent ciliary targeting mechanism, whereby Rabep1 couples the polycystin complex to a GGA1/Arl3-based ciliary trafficking module at the TGN.This study provides novel insights into the ciliary trafficking mechanism of membrane proteins.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

ABSTRACT
Primary cilia contain specific receptors and channel proteins that sense the extracellular milieu. Defective ciliary function causes ciliopathies such as autosomal dominant polycystic kidney disease (ADPKD). However, little is known about how large ciliary transmembrane proteins traffic to the cilia. Polycystin-1 (PC1) and -2 (PC2), the two ADPKD gene products, are large transmembrane proteins that co-localize to cilia where they act to control proper tubular diameter. Here we describe that PC1 and PC2 must interact and form a complex to reach the trans-Golgi network (TGN) for subsequent ciliary targeting. PC1 must also be proteolytically cleaved at a GPS site for this to occur. Using yeast two-hybrid screening coupled with a candidate approach, we identify a Rabep1/GGA1/Arl3-dependent ciliary targeting mechanism, whereby Rabep1 couples the polycystin complex to a GGA1/Arl3-based ciliary trafficking module at the TGN. This study provides novel insights into the ciliary trafficking mechanism of membrane proteins.

Show MeSH
Related in: MedlinePlus