The adaptor protein AP-4 as a component of the clathrin coat machinery: a morphological study

Abstract

The four members of the AP (adaptor protein) family are heterotetrameric cytosolic complexes that are involved in the intracellular trafficking of cargo proteins between different organelles. They interact with motifs present in the cytoplasmic tails of their specific cargo proteins at different intracellular locations. While AP-1, AP-2 and AP-3 have been investigated extensively, very few studies have focused on the fourth member, AP-4. In the present study, we report on the intracellular localization of AP-4 in the MDCK (Madin-Darby canine kidney) and MelJuSo cell lines after immunogold labelling of ultrathin cryosections. We find that AP-4 is localized mainly in the Golgi complex, as well as on endosomes and transport vesicles. Interestingly, we show for the first time that AP-4 is localized with the clathrin coat machinery in the Golgi complex and in the endocytic pathway. Furthermore, we find that AP-4 is localized with the CI-MPR (cation-independent mannose 6-phosphate receptor), but not with the transferrin receptor, LAMP-2 (lysosomal-associated membrane protein-2) or invariant chain. The difference in morphology between CI-MPR/AP-4-positive vesicles and CI-MPR/AP-1-positive vesicles raises the possibility that AP-4 acts at a location different from that of AP-1 in the intracellular trafficking pathway of CI-MPR.

Figure 1. Localization of AP-4 ε subunit in MDCK cells

Sections were labelled for the ε subunit of AP-4 (10 nm). Arrowheads indicate coated vesicles and membranes labelled for AP-4. Scale bar, 100 nm. G, Golgi complex; e, endosomes; n, nucleus; p, plasma membrane.

Figure 2. Localization of AP-4 μ subunit in MDCK cells

Sections were labelled for the μ subunit of AP-4 (10 nm). Arrowheads indicate coated vesicles labelled for AP-4. Arrows indicate vesicles bound to endosomes and labelled for AP-4. Scale bar, 100 nm. G, Golgi complex; e, endosomes.

Figure 3. Localization of AP-4 and clathrin in MDCK cells

Sections were double-labelled for the clathrin heavy chain (5 nm) and the μ subunit of AP-4 (15 nm). Arrows indicate the presence of AP-4 on the clathrin-coated membrane of endosomes. Scale bar, 100 nm. e, endosomes; m, mitochondria.

Figure 4. Localization of AP-4 in MelJuSo cells

Sections were labelled for the μ subunit of AP-4 (10 nm). In (B) and (C), endosomes contain internalized HRP (5 nm). The arrow indicates the coated membrane of an early endosome labelled for the μ4 subunit. Scale bar, 100 nm. G, Golgi complex; e, endosomes; p, plasma membrane.

Figure 5. Localization of AP-4 and different markers of the endocytic pathway in MelJuSo cells

Sections were double-labelled for the μ subunit of AP-4 (10 nm) plus (A) TfR (15 nm), (B) LAMP-2 (15 nm) or (C) Ii (15 nm). Scale bar, 100 nm. G, Golgi complex; e, endosomes; l, lysosomes; p, plasma membrane.

Figure 6. Localization of AP-4 and CI-MPR in MelJuSo cells

Sections were double-labelled for the μ subunit of AP-4 (10 nm) and CI-MPR (15 nm). Arrowheads indicate vesicles positive for AP-4 and CI-MPR. The arrow indicates the presence of AP-4 on the tip of a tubule emerging from an endosome. Scale bar, 100 nm. G, Golgi complex; e, endosomes.

Figure 7. Localization of AP-1 and CI-MPR in MelJuSo cells

Sections were double-labelled for the γ subunit of AP-1 (10 nm) and CI-MPR (15 nm). Arrowheads indicate vesicles positive for AP-1 and CI-MPR or AP-1 alone. Arrows indicate electron-dense vesicles labelled for CI-MPR only. Inset D′ shows an electron-dense vesicle positive for CI-MPR at 2× magnification; insert D″ shows a coated vesicle positive for AP-1 at 2× magnification. Scale bar, 100 nm. G, Golgi complex; e, endosomes; l, lysosomes; m, mitochondria; p, plasma membrane.