Synaptic vesicle recycling involves AP‐2/clathrin‐mediated endocytosis, but it is not known whether the endosomal pathway is also required. Mice deficient in the tissue‐specific AP‐1–σ1B complex have impaired synaptic vesicle recycling in hippocampal synapses. The ubiquitously expressed AP‐1–σ1A complex mediates protein sorting between the trans‐Golgi network and early endosomes. Vertebrates express three σ1 subunit isoforms: A, B and C. The expressions of σ1A and σ1B are highest in the brain. Synaptic vesicle reformation in cultured neurons from σ1B‐deficient mice is reduced upon stimulation, and large endosomal intermediates accumulate. The σ1B‐deficient mice have reduced motor coordination and severely impaired long‐term spatial memory. These data reveal a molecular mechanism for a severe human X‐chromosome‐linked mental retardation.

S U M M A R YWe have recently shown that monodansylcadaverine labels autophagic vacuoles. Analysis of the mechanism underlying the labeling revealed that monodansylcadaverine acts as a lysosomotropic agent, being concentrated into acidic compartments by an iontrapping mechanism, and as a solvent polarity probe, increasing its relative fluorescence intensity by interacting with membrane lipids that are highly concentrated in the autophagic vacuoles. In this study, we synthesized three structurally related derivatives of monodansylcadaverine, replacing the primary amino group of monodansylcadaverine with a neutral (dansylamylamine; MDH), a polar (dansylaminopentanol; MDOH), or an acidic group (dansylaminovaleric acid; MDA), to replace the lysosomotropic character of the marker. Whereas MDH showed a specific staining of autophagic vacuoles, the polar and acidic derivatives did not show any staining. We further demonstrate that the MDH staining of autophagic vacuoles is independent on the acidic pH and thus on an ion-trapping mechanism, but it still shows the same preferences for autophagic membrane lipids as monodansylcadaverine. We propose that MDH can specifically interact with lamellar bodies of the autophagic type as a solvent polarity probe. Therefore, dansylated aminopentane can be used as a specific marker for autophagic vacuoles in vivo and in fixed cells.

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SummaryThe AP-1 complex recycles between membranes and the cytoplasm and dissociates from membranes during clathrin-coated-vesicle uncoating, but also independently of vesicular transport. The m1A N-terminal 70 amino acids are involved in regulating AP-1 recycling. In a yeast two-hybrid library screen we identified the cytoplasmic prolyl-oligopeptidase-like protein PREPL as an interaction partner of this domain. PREPL overexpression leads to reduced AP-1 membrane binding, whereas reduced PREPL expression increases membrane binding and impairs AP-1 recycling. Altered AP-1 membrane binding in PREPL-deficient cells mirrors the membrane binding of the mutant AP-1* complex, which is not able to bind PREPL. Colocalisation of PREPL with residual membrane-bound AP-1 can be demonstrated. Patient cell lines deficient in PREPL have an expanded trans-Golgi network, which could be rescued by PREPL expression. These data demonstrate PREPL as an AP-1 effector that takes part in the regulation of AP-1 membrane binding. PREPL is highly expressed in brain and at lower levels in muscle and kidney. Its deficiency causes hypotonia and growth hormone hyposecretion, supporting essential PREPL functions in AP-1-dependent secretory pathways.