Jack Rohrer

ErbB-1 and ErbB-2 Acquire Distinct Signaling Properties Dependent upon Their Dimerization Partner

ABSTRACT The different epidermal growth factor (EGF)-related peptides elicit a diverse array of biological responses as the result of their ability to activate distinct subsets of ErbB receptor dimers, leading to the recruitment of different intracellular signaling networks. To specifically examine dimerization-dependent modulation of receptor signaling, we constructed NIH 3T3 cell lines expressing ErbB-1 and ErbB-2 singly and in pairwise combinations with each other ErbB family member. This model system allowed the comparison of EGF-activated ErbB-1 with ErbB-1 activated by Neu differentiation factor (NDF)-induced heterodimerization with ErbB-4. In both cases, ErbB-1 coupled to the adaptor protein Shc, but only when activated by EGF was it able to interact with Grb2. Compared to the rapid internalization of EGF-activated ErbB-1, NDF-activated ErbB-1 showed delayed internalization characteristics. Furthermore, the p85 subunit of phosphatidylinositol kinase (PI3-K) associated with EGF-activated ErbB-1 in a biphasic manner, whereas association with ErbB-1 transactivated by ErbB-4 was monophasic. The signaling properties of ErbB-2 following heterodimerization with the other ErbB receptors or homodimerization induced by point mutation or monoclonal antibody treatment were also analyzed. ErbB-2 binding to peptides containing the Src homology 2 domain of Grb2 or p85 and the phosphotyrosine binding domain of Shc varied according to the mode of receptor activation. Finally, tryptic phosphopeptide mapping of both ErbB-1 and ErbB-2 revealed that receptor phosphorylation is dependent on the dimerization partner. Differential receptor phosphorylation may, therefore, be the basis for the differences in the signaling properties observed.

Drosophila Vps16A is required for trafficking to lysosomes and biogenesis of pigment granules

Mutations that disrupt trafficking to lysosomes and lysosome-related organelles cause multiple diseases, including Hermansky-Pudlak syndrome. The Drosophila eye is a model system for analyzing such mutations. The eye-color genes carnation and deep orange encode two subunits of the Vps-C protein complex required for endosomal trafficking and pigment-granule biogenesis. Here we demonstrate that dVps16A (CG8454) encodes another Vps-C subunit. Biochemical experiments revealed a specific interaction between the dVps16A C-terminus and the Sec1/Munc18 homolog Carnation but not its closest homolog, dVps33B. Instead, dVps33B interacted with a related protein, dVps16B (CG18112). Deep orange bound both Vps16 homologs. Like a deep orange null mutation, eye-specific RNAi-induced knockdown of dVps16A inhibited lysosomal delivery of internalized ligands and interfered with biogenesis of pigment granules. Ubiquitous knockdown of dVps16A was lethal. Together, these findings demonstrate that Drosophila Vps16A is essential for lysosomal trafficking. Furthermore, metazoans have two types of Vps-C complexes with non-redundant functions.

Alzheimer's Disease-related Overexpression of the Cation-dependent Mannose 6-Phosphate Receptor Increases Aβ Secretion ROLE FOR ALTERED LYSOSOMAL HYDROLASE DISTRIBUTION IN β-AMYLOIDOGENESIS

Prominent endosomal and lysosomal changes are an invariant feature of neurons in sporadic Alzheimers disease (AD). These changes include increased levels of lysosomal hydrolases in early endosomes and increased expression of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is partially localized to early endosomes. To determine whether AD-associated redistribution of lysosomal hydrolases resulting from changes in CD-MPR expression affects amyloid precursor protein (APP) processing, we stably transfected APP-overexpressing murine L cells with human CD-MPR. As controls for these cells, we also expressed CD-MPR trafficking mutants that either localize to the plasma membrane (CD-MPRpm) or to early endosomes (CD-MPRendo). Expression of CD-MPR resulted in a partial redistribution of a representative lysosomal hydrolase, cathepsin D, to early endosomal compartments. Turnover of APP and secretion of sAPPα and sAPPβ were not altered by overexpression of any of the CD-MPR constructs. However, secretion of both human Aβ40 and Aβ42 into the growth media nearly tripled in CD-MPR- and CD-MPRendo-expressing cells when compared with parental or CD-MPRpm-expressing cells. Comparable increases were confirmed for endogenous mouse Aβ40 in L cells expressing these CD-MPR constructs but not overexpressing human APP. These data suggest that redistribution of lysosomal hydrolases to early endocytic compartments mediated by increased expression of the CD-MPR may represent a potentially pathogenic mechanism for accelerating Aβ generation in sporadic AD, where the mechanism of amyloidogenesis is unknown.

Neonatal lethality in mice deficient in XCE, a novel member of the endothelin-converting enzyme and neutral endopeptidase family

XCE, a new member of the endothelin-converting enzyme and neutral endopeptidase family, is preferentially expressed in specific areas of the central nervous system including spinal chord and medulla. To elucidate the importance and function of XCE, we disrupted its gene in mouse embryonic stem cells by homologous recombination and created mice deficient in XCE. The resulting phenotype is characterized by neonatal lethality. All XCE −/− homozygous mice died of respiratory failure shortly after birth, and in most cases their lungs were never ventilated. Apart from the atelectasis, anatomical and histological examinations of embryonic day 18.5 XCE −/− embryos and newborn homozygotes did not reveal any obvious abnormalities in organs and tissues. Malformations that are related to the knock-out were also not found in the skeletons of XCE −/− mice. In addition, XCE knock-out animals showed no deficiency of pulmonary surfactant proteins and had normal heart beat frequencies. Taken together, our results demonstrate that XCE is an essential gene. The phenotype of the XCE-deficient mice together with the central nervous system-specific expression further suggest that XCE may play a vital role in the control of respiration.

A di-aromatic motif in the cytosolic tail of the mannose receptor mediates endosomal sorting.

The mannose receptor (MR), the prototype of a new family of multilectin receptor proteins important in innate immunity, undergoes rapid internalization and recycling from the endosomal system back to the cell surface. Sorting of the MR in endosomes prevents the receptor from entering lysosomes where it would be degraded. Here, we focused on a diaromatic sequence (Tyr18-Phe19) in the MR cytoplasmic tail as an endosomal sorting signal. The subcellular distribution of chimeric constructs between the MR and the cation-dependent mannose 6-phosphate receptor was assessed by Percoll density gradients and cell surface assays. Unlike the wild type constructs, mutant receptors with alanine substitutions of Tyr18-Phe19 were highly missorted to lysosomes, indicating that the di-aromatic motif of the MR cytoplasmic tail mediates sorting in endosomes. Within this sequence Tyr18is the key residue with Phe19 contributing to this function. Moreover, Tyr18 was also found to be essential for internalization, consistent with the presence of overlapping signals for internalization and endosomal sorting in the cytosolic tail of the MR. A di-aromatic amino acid sequence in the cytosolic tail has now been shown to function in two receptors known to be internalized from the plasma membrane, the MR and the cation-dependent mannose 6-phosphate receptor. This feature therefore appears to be a general determinant for endosomal sorting.

Differential effects of lobe A and lobe B of the Conserved Oligomeric Golgi complex on the stability of β1,4-galactosyltransferase 1 and α2,6-sialyltransferase 1

Initially described by Jaeken et al. in 1980, congenital disorders of glycosylation (CDG) is a rapidly expanding group of human multisystemic disorders. To date, many CDG patients have been identified with deficiencies in the conserved oligomeric Golgi (COG) complex which is a complex involved in the vesicular intra-Golgi retrograde trafficking. Composed of eight subunits that are organized in two lobes, COG subunit deficiencies have been associated with Golgi glycosylation abnormalities. Analysis of the total serum N-glycans of COG-deficient CDG patients demonstrated an overall decrease in terminal sialylation and galactosylation. According to the mutated COG subunits, differences in late Golgi glycosylation were observed and led us to address the question of an independent role and requirement for each of the two lobes of the COG complex in the stability and localization of late terminal Golgi glycosylation enzymes. For this, we used a small-interfering RNAs strategy in HeLa cells stably expressing green fluorescent protein (GFP)-tagged β1,4-galactosyltransferase 1 (B4GALT1) and α2,6-sialyltransferase 1 (ST6GAL1), two major Golgi glycosyltransferases involved in late Golgi N-glycosylation. Using fluorescent lectins and flow cytometry analysis, we clearly demonstrated that depletion of both lobes was associated with deficiencies in terminal Golgi N-glycosylation. Lobe A depletion resulted in dramatic changes in the Golgi structure, whereas lobe B depletion severely altered the stability of B4GALT1 and ST6GAL1. Only MG132 was able to rescue their steady-state levels, suggesting that B4GALT1- and ST6GAL1-induced degradation are likely the consequence of an accumulation in the endoplasmic reticulum (ER), followed by a retrotranslocation into the cytosol and proteasomal degradation. All together, our results suggest differential effects of lobe A and lobe B for the localization/stability of B4GALT1 and ST6GAL1. Lobe B would be crucial in preventing these two Golgi glycosyltransferases from inappropriate retrograde trafficking to the ER, whereas lobe A appears to be essential for maintaining the overall Golgi structure.

Galactosyltransferase—still up and running☆

The following review on galactosyltransferase (gal-T1) intends to cover genetic, biochemical, structural, biotechnological, cell biological and medical aspects of this enzyme in a comprehensive manner from discovery to the present day which have brought to light a genetic defect of this enzyme. Early work has only been included if it appeared relevant to ongoing issues. Following the evolution of a research topic over 40 years is in itself a fascinating endeavor as it permits to observe the ins and outs of hypotheses, fashions and errors. Gal-T1 is a beautiful example as it has been involved in almost every aspect of life science. Importantly, there is a future to this enzyme as a research topic, since many questions still remain unanswered: to which extent is it a representative Golgi protein? What is the role of the gene family of gal-Ts? Does gal-T1 exert any functions other than a catalytic one? Why is it phosphorylated? Does it form homodimers in vivo? Surely, there is room for further work, which is likely to reveal further insights into cellular trafficking and signaling and, in the context of the gene family, shall contribute to understanding development and morphogenesis.

Characterization of the TGN exit signal of the human mannose 6-phosphate uncovering enzyme

The human mannose 6-phosphate uncovering enzyme participates in the uncovering of the mannose 6-phosphate recognition tag on lysosomal enzymes, a process that facilitates recognition of those enzymes by mannose 6-phosphate receptors to ensure delivery to lysosomes. Uncovering enzyme has been identified on the trans-Golgi network at steady state. It has been shown to traffic to the plasma membrane from where it is rapidly internalized via endosomal structures, the process being mediated by a tyrosine-based internalization motif, Y488HPL, in its cytoplasmic tail. Using immunogold electron microscopy a GFP-uncovering enzyme fusion construct was found to be colocalized with the cation-dependent mannose 6-phosophate receptor in regions of the trans-Golgi network, suggesting that uncovering enzyme might follow a similar pathway of exit from the trans-Golgi network as that of the cation-dependent mannose 6-phosohate receptor. In this study, we identified the signal sequence in the cytoplasmic tail of uncovering enzyme responsible for its exit from the trans-Golgi network. Using GFP fusion constructs of the transmembrane and cytoplasmic domains of uncovering enzyme, we could show, by automated analysis of confocal immunofluorescence images, that residues Q492EMN in the cytoplasmic tail of uncovering enzyme are involved in its exit from the trans-Golgi network. Detailed characterization of the exit signal revealed that residue Q492 is the most important to the exit function while M494 and N495 also contribute. The cytoplasmic tail of the uncovering enzyme does not possess any of the known canonical signal sequences for interaction with Golgi-associated gamma ear-containing adaptor proteins. The identification of a trans-Golgi network exit signal in its cytoplasmic tail elucidates the trafficking pathway of uncovering enzyme, a crucial player in the process of lysosomal biogenesis.

Analysis of protein transport to lysosomes

Lysosomes are terminal degradative organelles that are found in all higher eukaryotic cells. The biogenesis of lysosomes involves the transport of various acid hydrolases and transmembrane glycoproteins from their site of synthesis in the endoplasmic reticulum through the biosynthetic and endocytic pathways. Protein transport to lysosomes can be studied by a combination of techniques based on the separation of intracellular organelles. Percoll density gradient centrifugation has long been the method of choice for separating lysosomes from other organelles in cell homogenates, and accordingly, this unit describes protocols for obtaining reasonably pure lysosomal fractions from mammalian cells using Percoll density gradient separation.

Dissection of a novel molecular determinant mediating Golgi to trans -Golgi network transition

Abstract.Two major functions of the Golgi apparatus (GA) are formation of complex glycans and sorting of proteins destined for various subcellular compartments or secretion. To fulfill these tasks proper localization of the accessory proteins within the different sub-compartments of the GA is crucial. Here we investigate structural determinants mediating transition of the two glycosyltransferases β-1,4- galactosyltransferase 1 (gal-T1) and the α-1,3-fucosyltransferase 6 (fuc-T6) from the trans-Golgi cisterna to the trans-Golgi network (TGN). Upon treatment with the ionophore monensin both glycosyltransferases are found in TGN-derived swollen vesicles, as determined by confocal fluorescence microscopy and density gradient fractionation. Both enzymes carry a signal consisting of the amino acids E5P6 in gal-T1 and D2P3 in fuc-T6 necessary for the transition of these glycosyltransferases from the trans-Golgi cisterna to the TGN, but not for their steady state localization in the trans-Golgi cisterna.