One of the hallmarks of Alzheimer\'s disease is the accumulation of toxic amyloid-β (Aβ) peptides in extracellular plaques. The direct precursor of Aβ is the carboxyl-terminal fragment β (or C99) of the amyloid precursor protein (APP). C99 is detected at elevated levels in Alzheimer\'s disease brains, and its intracellular accumulation has been linked to early neurotoxicity independently of Aβ. Despite this, the causes of increased C99 levels are poorly understood. Here, we demonstrate that APP interacts with the clathrin vesicle adaptor AP-1 (adaptor protein 1), and we map the interaction sites on both proteins. Using quantitative kinetic trafficking assays, established cell lines and primary neurons, we also show that this interaction is required for the transport of APP from the trans-Golgi network to endosomes. In addition, disrupting AP-1-mediated transport of APP alters APP processing and degradation, ultimately leading to increased C99 production and Aβ release. Our results indicate that AP-1 regulates the subcellular distribution of APP, altering its processing into neurotoxic fragments.

Most proteins in the secretory pathway are glycosylated. However, the role of glycans in membrane trafficking is still unclear. Here, we discovered that transmembrane secretory cargos, such as interleukin 2 receptor α subunit or Tac, transferrin receptor, and cluster of differentiation 8a, unexpectedly displayed substantial Golgi localization when their O-glycosylation was compromised. By quantitatively measuring their Golgi residence times, we found that the observed Golgi localization of O-glycan-deficient cargos is due to their slow Golgi export. Using a superresolution microscopy method that we previously developed, we revealed that O-glycan-deficient Tac chimeras localize at the interior of the trans-Golgi cisternae. O-Glycans were observed to be both necessary and sufficient for the efficient Golgi export of Tac chimeras. By sequentially introducing O-glycosylation sites to ST6GAL1, we demonstrated that O-glycan\'s effect on Golgi export is probably additive. Finally, the finding that N-glycosylated GFP substantially reduces the Golgi residence time of a Tac chimera suggests that N-glycans might have a similar effect. Therefore, both O- and N-glycans might function as a generic Golgi export signal at the trans-Golgi to promote the constitutive exocytic trafficking.

The voltage-gated sodium channel is critical for cardiomyocyte function and consists of a protein complex comprising a pore-forming α subunit and two associated β subunits. It has been shown previously that the associated β2 subunits promote cell surface expression of the α subunit. The major α isoform in the adult human heart is NaV1.5, and germline mutations in the NaV1.5-encoding gene, sodium voltage-gated channel α subunit 5 (SCN5A), often cause inherited arrhythmias. Here, we investigated the mechanisms that regulate β2 trafficking and how they may determine proper NaV1.5 cell surface localization. Using heterologous expression in polarized Madin-Darby canine kidney cells, we show that β2 is N-glycosylated in vivo and in vitro at residues 42, 66, and 74, becoming sialylated only at Asn-42. We found that fully nonglycosylated β2 was mostly retained in the endoplasmic reticulum, indicating that N-linked glycosylation is required for efficient β2 trafficking to the apical plasma membrane. The nonglycosylated variant reached the cell surface by bypassing the Golgi compartment at a rate of only approximately one-third of that of WT β2. YFP-tagged, nonglycosylated β2 displayed mobility kinetics in the plane of the membrane similar to that of WT β2. However, it was defective in promoting surface localization of NaV1.5. Interestingly, β2 with a single intact glycosylation site was as effective as the WT in promoting NaV1.5 surface localization. In conclusion, our results indicate that N-linked glycosylation of β2 is required for surface localization of NaV1.5, a property that is often defective in inherited cardiac arrhythmias.

The architecture and organization of the Golgi complex depend on a family of coiled-coil proteins called golgins. Golgins are thought to form extended homodimers that are C-terminally anchored to Golgi membranes, whereas their N termini extend into the cytoplasm to initiate vesicle capture. Previously, we reported that the Saccharomyces cerevisiae golgin Coy1 contributes to intra-Golgi retrograde transport and binds to the conserved oligomeric Golgi (COG) complex and multiple retrograde Golgi Q-SNAREs (where SNARE is soluble NSF-attachment protein receptor). Here, using various engineered yeast strains, membrane protein extraction and fractionation methods, and in vitro binding assays, we mapped the Coy1 regions responsible for these activities. We also report that Coy1 assembles into a megadalton-size complex and that assembly of this complex depends on the most C-terminal coiled-coil and a conserved region between this coiled-coil and the transmembrane domain of Coy1. We found that this conserved region is necessary and sufficient for binding the SNARE protein Sed5 and the COG complex. Mutagenesis of conserved arginine residues within the C-terminal coiled-coil disrupted oligomerization, binding, and function of Coy1. Our findings indicate that the stable incorporation of Coy1 into a higher-order oligomer is required for its interactions and role in maintaining Golgi homeostasis. We propose that Coy1 assembles into a docking platform that directs COG-bound vesicles toward cognate SNAREs on the Golgi membrane.

Amyloid precursor protein (APP) is processed along the amyloidogenic pathway by the β-secretase, BACE1, generating β-amyloid (Aβ), or along the nonamyloidogenic pathway by α-secretase, precluding Aβ production. The plasma membrane is considered the major site for α-secretase-mediated APP cleavage, but other cellular locations have not been rigorously investigated. Here, we report that APP is processed by endogenous α-secretase at the trans-Golgi network (TGN) of both transfected HeLa cells and mouse primary neurons. We have previously shown the adaptor protein complex, AP-4, and small G protein ADP-ribosylation factor-like GTPase 5b (Arl5b) are required for efficient post-Golgi transport of APP to endosomes. We found here that AP-4 or Arl5b depletion results in Golgi accumulation of APP and increased secretion of the soluble α-secretase cleavage product sAPPα. Moreover, inhibition of γ-secretase following APP accumulation in the TGN increases the levels of the membrane-bound C-terminal fragments of APP from both α-secretase cleavage (α-CTF, named C83 according to its band size) and BACE1 cleavage (β-CTF/C99). The level of C83 was ∼4 times higher than that of C99, indicating that α-secretase processing is the major pathway and that BACE1 processing is the minor pathway in the TGN. AP-4 silencing in mouse primary neurons also resulted in the accumulation of endogenous APP in the TGN and enhanced α-secretase processing. These findings identify the TGN as a major site for α-secretase processing in HeLa cells and primary neurons and indicate that both APP processing pathways can occur within the TGN compartment along the secretory pathway.

Cellular membrane trafficking mediated by the clathrin adaptor protein complex-1 (AP-1) is important for the proper composition and function of organelles of the endolysosomal system. Normal AP-1 function requires proteins of the HEAT repeat-containing 5 (HEATR5) family. Although HEATR5 proteins were first identified based on their ability to interact with AP-1, the functional significance of this interaction was unknown. We used bioinformatics-based phenotypic profiling and information from genome-wide fluorescence microscopy studies in the budding yeast Saccharomyces cerevisiae to identify a protein, Laa2, that mediates the interaction between AP-1 and the yeast HEATR5 protein Laa1. Further characterization of Laa2 revealed that it binds to both Laa1 and AP-1. Laa2 contains a motif similar to the characterized γ-ear-binding sites found in other AP-1-binding proteins. This motif in Laa2 is essential for the Laa1-AP-1 interaction. Moreover, mutation of this motif disrupted AP-1 localization and function and caused effects similar to mutations that remove the γ-ear of AP-1. These results indicate that Laa2 mediates the interaction between Laa1 and AP-1 and reveal that this interaction promotes the stable association of AP-1 with membranes in yeast.

Human myeloid-derived growth factor (MYDGF; also known as C19orf10) is named based on its identification as a secreted monocyte/macrophage-derived mediator of cardiac repair following myocardial infarction in mice. Homologs of MYDGF, however, are present in organisms throughout and outside of the animal kingdom, some of which lack hematopoietic and circulatory systems. Moreover, the UPF0556 protein domain, which defines these homologs, lacks a known structure. As a result, the functions and properties of MYDGF are unclear. Our current work was initiated to test whether MYDGF is present in secretory vesicles of eosinophils as it was recently reported to be abundant in these cells. However, we could not demonstrate secretion and unexpectedly discovered that MYDGF colocalizes with P4HB in the nuclear envelope, which comprises the bulk of endoplasmic reticulum (ER) in eosinophils, and with P4HB and RCAS1 in Golgi. We noted a ubiquitous C-terminal sequence, BXEL (B, basic; X, variable residue; E, Glu; L, Leu), that has the potential to retain human MYDGF and its homologs in the ER. To test the functionality of this sequence, we expressed full-length human MYDGF or MYDGF lacking the C-terminal Glu-Leu residues in monolayers of human embryonic kidney 293 (HEK293) cells. Full-length MYDGF accumulated in cells, whereas truncated MYDGF appeared in the medium. These observations reveal that MYDGF resides in the ER and Golgi and provide a new framework for investigating and understanding this intriguing protein.

Protein composition at the plasma membrane is tightly regulated, with rapid protein internalization and selective targeting to the cell surface occurring in response to environmental changes. For example, ion channels are dynamically relocalized to or from the plasma membrane in response to physiological alterations, allowing cells and organisms to maintain osmotic and salt homeostasis. To identify additional factors that regulate the selective trafficking of a specific ion channel, we used a yeast model for a mammalian potassium channel, the K+ inward rectifying channel Kir2.1. Kir2.1 maintains potassium homeostasis in heart muscle cells, and Kir2.1 defects lead to human disease. By examining the ability of Kir2.1 to rescue the growth of yeast cells lacking endogenous potassium channels, we discovered that specific α-arrestins regulate Kir2.1 localization. Specifically, we found that the Ldb19/Art1, Aly1/Art6, and Aly2/Art3 α-arrestin adaptor proteins promote Kir2.1 trafficking to the cell surface, increase Kir2.1 activity at the plasma membrane, and raise intracellular potassium levels. To better quantify the intracellular and cell-surface populations of Kir2.1, we created fluorogen-activating protein fusions and for the first time used this technique to measure the cell-surface residency of a plasma membrane protein in yeast. Our experiments revealed that two α-arrestin effectors also control Kir2.1 localization. In particular, both the Rsp5 ubiquitin ligase and the protein phosphatase calcineurin facilitated the α-arrestin-mediated trafficking of Kir2.1. Together, our findings implicate α-arrestins in regulating an additional class of plasma membrane proteins and establish a new tool for dissecting the trafficking itinerary of any membrane protein in yeast.

In planar cell polarity (PCP), the epithelial cells are polarized along the plane of the cell surface perpendicular to the classical apical-basal axis, a process mediated by several conserved signaling receptors. Two PCP-signaling proteins, VANGL planar cell polarity protein 2 (Vangl2) and Frizzled6 (Fzd6), are located asymmetrically on opposite boundaries of the cell. Examining sorting of these two proteins at the trans-Golgi network (TGN), we demonstrated previously that the GTP-binding protein ADP-ribosylation factor-related protein 1 (Arfrp1) and the clathrin-associated adaptor protein complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled6 does not depend on Arfrp1 or AP-1. Here, to further investigate the TGN sorting process in mammalian cells, we reconstituted release of Vangl2 and Frizzled6 from the TGN into vesicles in vitro Immunoblotting of released vesicles indicated that Vangl2 and Frizzled6 exit the TGN in separate compartments. Knockdown analysis revealed that a clathrin adaptor, epsinR, regulates TGN export of Frizzled6 but not of Vangl2. Protein interaction analysis suggested that epsinR forms a stable complex with clathrin and that this complex interacts with a conserved polybasic motif in the Frizzled6 cytosolic domain to package Frizzled6 into transport vesicles. Moreover, we found that Frizzled6-epsinR binding dissociates epsinR from AP-1, which may separate these two cargo adaptors from each other to perform distinct cargo-sorting functions. Our results suggest that Vangl2 and Frizzled6 are packaged into separate vesicles that are regulated by different clathrin adaptors at the TGN, which may contribute to their asymmetric localizations.

An endoplasmic reticulum (ER) retention sequence (ERS) is a characteristic short sequence that mediates protein retention in the ER of eukaryotic cells. However, little is known about the detailed molecular mechanism involved in ERS-mediated protein ER retention. Using a new surface display-based fluorescence technique that effectively quantifies ERS-promoted protein ER retention within Saccharomyces cerevisiae cells, we performed comprehensive ERS analyses. We found that the length, type of amino acid residue, and additional residues at positions -5 and -6 of the C-terminal HDEL motif all determined the retention of ERS in the yeast ER. Moreover, the biochemical results guided by structure simulation revealed that aromatic residues (Phe-54, Trp-56, and other aromatic residues facing the ER lumen) in both the ERS (at positions -6 and -4) and its receptor, Erd2, jointly determined their interaction with each other. Our studies also revealed that this aromatic residue interaction might lead to the discriminative recognition of HDEL or KDEL as ERS in yeast or human cells, respectively. Our findings expand the understanding of ERS-mediated residence of proteins in the ER and may guide future research into protein folding, modification, and translocation affected by ER retention.