Peter Schu

μ1A‐adaptin‐deficient mice: lethality, loss of AP‐1 binding and rerouting of mannose 6‐phosphate receptors

The heterotetrameric AP‐1 complex is involved in the formation of clathrin‐coated vesicles at the trans‐Golgi network (TGN) and interacts with sorting signals in the cytoplasmic tails of cargo molecules. Targeted disruption of the mouse μ1A‐adaptin gene causes embryonic lethality at day 13.5. In cells deficient in μ1A‐adaptin the remaining AP‐1 adaptins do not bind to the TGN. Polarized epithelial cells are the only cells of μ1A‐adaptin‐deficient embryos that show γ‐adaptin binding to membranes, indicating the formation of an epithelial specific AP‐1B complex and demonstrating the absence of additional μ1A homologs. Mannose 6‐phosphate receptors are cargo molecules that exit the TGN via AP‐1–clathrin‐coated vesicles. The steady‐state distribution of the mannose 6‐phosphate receptors MPR46 and MPR300 in μ1A‐deficient cells is shifted to endosomes at the expense of the TGN. MPR46 fails to recycle back from the endosome to the TGN, indicating that AP‐1 is required for retrograde endosome to TGN transport of the receptor.

Clathrin Adaptor epsinR Is Required for Retrograde Sorting on Early Endosomal Membranes

Retrograde transport links early/recycling endosomes to the trans-Golgi network (TGN), thereby connecting the endocytic and the biosynthetic/secretory pathways. To determine how internalized molecules are targeted to the retrograde route, we have interfered with the function of clathrin and that of two proteins that interact with it, AP1 and epsinR. We found that the glycosphingolipid binding bacterial Shiga toxin entered cells efficiently when clathrin expression was inhibited. However, retrograde transport of Shiga toxin to the TGN was strongly inhibited. This allowed us to show that for Shiga toxin, retrograde sorting on early/recycling endosomes depends on clathrin and epsinR, but not AP1. EpsinR was also involved in retrograde transport of two endogenous proteins, TGN38/46 and mannose 6-phosphate receptor. In conclusion, our work reveals the existence of clathrin-independent and -dependent transport steps in the retrograde route, and establishes a function for clathrin and epsinR at the endosome-TGN interface.

Sorting by the Cytoplasmic Domain of the Amyloid Precursor Protein Binding Receptor SorLA

ABSTRACT SorLA/LR11 (250 kDa) is the largest and most composite member of the Vps10p-domain receptors, a family of type 1 proteins preferentially expressed in neuronal tissue. SorLA binds several ligands, including neurotensin, platelet-derived growth factor-bb, and lipoprotein lipase, and via complex-formation with the amyloid precursor protein it downregulates generation of Alzheimers disease-associated Aβ-peptide. The receptor is mainly located in vesicles, suggesting a function in protein sorting and transport. Here we examined SorLAs trafficking using full-length and chimeric receptors and find that its cytoplasmic tail mediates efficient Golgi body-endosome transport, as well as AP-2 complex-dependent endocytosis. Functional sorting sites were mapped to an acidic cluster-dileucine-like motif and to a GGA binding site in the C terminus. Experiments in permanently or transiently AP-1 μ1-chain-deficient cells established that the AP-1 adaptor complex is essential to SorLAs transport between Golgi membranes and endosomes. Our results further implicate the GGA proteins in SorLA trafficking and provide evidence that SNX1 and Vps35, as parts of the retromer complex or possibly in a separate context, are engaged in retraction of the receptor from endosomes.

Early embryonic death of mice deficient in gamma-adaptin

Intracellular protein transport and sorting by vesicles in the secretory and endocytic pathways requires the formation of a protein coat on the membrane. The heterotetrameric adaptor protein complex 1 (AP-1) promotes the formation of clathrin-coated vesicles at the trans-Golgi network. AP-1 interacts with various sorting signals in the cytoplasmic tails of cargo molecules, thus indicating a function in protein sorting. We generated mutants of the γ-adaptin subunit of AP-1 in mice to investigate its role in post-Golgi vesicle transport and sorting processes. γ-Adaptin-deficient embryos develop until day 3.5 post coitus and die during the prenidation period, revealing that AP-1 is essential for viability. In heterozygous mice the amount of AP-1 complexes is reduced to half of controls. Free β1- or μ1 chains were not detectable, indicating that they are unstable unless they are part of AP-1 complexes. Heterozygous mice weigh less then their wild-type littermates and show impaired T cell development.

AP‐1/σ1B‐adaptin mediates endosomal synaptic vesicle recycling, learning and memory

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.

Rabaptin‐5α/rabaptin‐4 serves as a linker between rab4 and γ1‐adaptin in membrane recycling from endosomes

Rab4 regulates recycling from early endosomes. We investigated the role of the rab4 effector rabaptin‐5α and its putative partner γ1‐adaptin in membrane recycling. We found that rabaptin‐5α forms a ternary complex with the γ1–σ1 subcomplex of AP‐1, via a direct interaction with the γ1‐subunit. The binding site for γ1‐adaptin is in the hinge region of rabaptin‐5α, which is distinct from rab4‐ and rab5‐binding domains. Endogenous or ectopically expressed γ1‐ adaptin localized to both the trans‐Golgi network and endosomes. Co‐expressed rabaptin‐5α and γ1‐adaptin, however, co‐localized in a rab4‐dependent manner on recycling endosomes. Transfection of rabaptin‐5α caused enlarged endosomes and delayed recycling of transferrin. RNAi of rab4 had an opposing effect on transferrin recycling. Collectively, our data show that rab4‐GTP acts as a scaffold for a rabaptin‐5α–γ1‐adaptin complex on recycling endosomes and that interactions between rab4, rabaptin‐5α and γ1‐adaptin regulate membrane recycling.

AP-1A and AP-3A Lysosomal Sorting Functions

Heterotetrameric adaptor‐protein complexes AP‐1A and AP‐3A mediate protein sorting in post‐Golgi vesicular transport. AP‐1A and AP‐3A have been localized to the trans‐Golgi network, indicating a function in protein sorting at this compartment. AP‐3A appears to mediate trans‐Golgi network‐to‐lysosome and also endosome‐to‐lysosome protein sorting. AP‐1A is thought to be required for both trans‐Golgi network‐to‐endosome transport and endosome‐to‐trans‐Golgi network transport. However, the recent discovery of a role for monomeric GGA (Golgi localized γ‐ear containing, ARF binding protein) adaptor proteins in trans‐Golgi network to endosome protein transport has brought into question the long‐discussed trans‐Golgi network‐to‐endosome sorting function of AP‐1A. Murine cytomegalovirus gp48 contains an unusual di‐leucine‐based lysosome sorting signal motif and mediates lysosomal sorting of gp48/major histocompatibility complex class I receptor complexes, preventing exposure of major histocompatibility complex class I at the plasma membrane. We analyzed lysosomal sorting of gp48/major histocompatibility complex class I receptor complexes in cell lines deficient for AP‐1A, AP‐3A and both, to determine their sorting functions. We find that AP1‐A and AP3‐A mediate distinct and sequential steps in the lysosomal sorting. Both sorting functions are required to prevent MHC class I exposure at the plasma membrane at steady‐state.

Characterization of the in Vitro Retrograde Transport of MPR46

The mannose 6‐phosphate receptor MPR46 mediates sorting of lysosomal enzymes and recycles between the trans‐Golgi network and endosomes. We characterized the retrograde transport of MPR46 from endosomes to the TGN by an in vitro transport assay using mouse fibroblast cell lines. Sulfation of a modified MPR46 upon entering the TGN is measured. The in vitro retrograde transport is time‐, temperature‐, ATP‐ and cytosol‐dependent. Transport requires the SNARE proteins Vti1a and Syntaxin 16 and the Rab family member Rab6. The transport is sensitive to GTPγS, brefeldin A and independent of TIP47. These data indicate that MPR46 follows an early endosome‐to‐TGN route. Transport is inhibited by MPR46 tail peptide comprising the acidic cluster‐di‐leucine sorting motif to which adaptor proteins AP‐1 and AP‐3 bind. Transport depends on cytosolic AP‐3, but not on cytosolic AP‐1. Residual membrane‐associated AP‐1 may have masked a requirement for cytosolic AP‐1. The competence of membranes from AP‐1‐deficient cells for endosome‐to‐TGN transport in vitro was severely compromised.

γ-BAR, a novel AP-1-interacting protein involved in post-Golgi trafficking

A novel peripheral membrane protein (2c18) that interacts directly with the gamma ‘ear’ domain of the adaptor protein complex 1 (AP‐1) in vitro and in vivo is described. Ultrastructural analysis demonstrates a colocalization of 2c18 and γ1‐adaptin at the trans‐Golgi network (TGN) and on vesicular profiles. Overexpression of 2c18 increases the fraction of membrane‐bound γ1‐adaptin and inhibits its release from membranes in response to brefeldin A. Knockdown of 2c18 reduces the steady‐state levels of γ1‐adaptin on membranes. Overexpression or downregulation of 2c18 leads to an increased secretion of the lysosomal hydrolase cathepsin D, which is sorted by the mannose‐6‐phosphate receptor at the TGN, which itself involves AP‐1 function for trafficking between the TGN and endosomes. This suggests that the direct interaction of 2c18 and γ1‐adaptin is crucial for membrane association and thus the function of the AP‐1 complex in living cells. We propose to name this protein γ‐BAR.

Disruption of adaptor protein 2μ (AP-2μ) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing.

Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2μ (AP‐2μ) is required for release site replenishment and hearing. We show that hair cell‐specific disruption of AP‐2μ slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane‐proximal vesicles and intact endocytic membrane retrieval. Sound‐driven postsynaptic spiking was reduced in a use‐dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome‐like vacuoles, fewer clathrin‐coated endocytic intermediates, and vesicle depletion of the membrane‐distal synaptic ribbon in AP‐2μ‐deficient IHCs, indicating a further role of AP‐2μ in clathrin‐dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP‐2 sorts its IHC‐cargo otoferlin. We propose that binding of AP‐2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP‐2 in synaptic vesicle reformation.