Activation of the Endosome-Associated Ubiquitin Isopeptidase AMSH by STAM, a Component of the Multivesicular Body-Sorting Machinery

AMSH is an endosomal ubiquitin isopeptidase that can limit EGF receptor downregulation [1]. It directly binds to the SH3 domain of STAM, which is constitutively associated with Hrs, a component of clathrin-coated structures on endosomes. This clathrin coat has been implicated in the recruitment of ubiquitinated growth factor receptors prior to their incorporation into internal vesicles of the multivesicular body (MVB) 2 and 3, through the concerted action of ESCRT complexes I, II, and III [4]. We now show that AMSH is embedded within a network of interactions with components of the MVB-sorting machinery. AMSH and STAM, like Hrs [5], both bind directly to clathrin. AMSH also interacts with mVps24/CHMP3, a component of ESCRT III complex, and this interaction is reinforced through simultaneous STAM binding. We have explored the effect of interacting components on the in vitro enzymatic activity of AMSH. The enzyme shows specificity for K63- over K48-linked polyubiquitin chains in vitro and is markedly stimulated by coincubation with STAM, indicating that activation of AMSH is coupled to its association with the MVB-sorting machinery. Other interacting factors do not directly stimulate AMSH but may serve to orient the enzyme with respect to substrates on the endosomal membrane

The UIM domain of Hrs couples receptor sorting to vesicle formation

Hepatocyte growth factor regulated tyrosine kinase substrate (Hrs), a main component of the 'bilayered' clathrin coat on sorting endosomes, was originally identified as a substrate of activated tyrosine kinase receptors. We have analysed Hrs phosphorylation in response to epidermal growth factor (EGF) stimulation and show that the evolutionary conserved tyrosines Y329 and Y334 provide the principal phosphorylation sites. Hrs is proposed to concentrate ubiquitinated receptors within clathrin-coated regions via direct interaction with its UIM (ubiquitin interaction motif) domain. We show that the same UIM domain is necessary for EGF-stimulated tyrosine phosphorylation of Hrs. Over-expression of wild-type Hrs or a double mutant, Y329/334F, defective in EGF-dependent phosphorylation, both substantially retard EGF receptor (EGFR) degradation by inhibiting internal vesicle formation and thereby preventing EGFR incorporation into lumenal vesicles of the multivesicular bodies. In contrast, mutation or deletion of the Hrs-UIM domain strongly suppresses this effect. In addition the UIM-deletion and point mutants are also observed on internal membranes, indicating a failure to dissociate from the endosomal membrane prior to incorporation of the receptor complex into lumenal vesicles. Our data suggest a role for the UIM-domain of Hrs in actively retaining EGFR at the limiting membrane of endosomes as a prelude to lumenal vesicle formation

Report on the Forest Sector Project Network Meeting: 29 August - 2 September 1983 - Sopron, Hungary

As at the two previous network meetings, the 1983 Forest Sector Project Network Meeting summarized the accomplishments of the Project, both by the staff at IIASA in Laxenburg, Austria, and by the national teams in collaborating countries. Also discussed were options for future effort (particularly for further development of the global forest trade model), data required to be assembled and analyzed, and other Forest Sector Project affairs. A detailed agenda is listed in Appendix A. The network meeting was attended by 39 participants from 13 of the collaborating countries and from cooperating organizations (Food and Agriculture Organization of United Nations (FAO), United Nations Industrial Development Organization (UNIDO), and Jaakko Poyry International Oy). Eight members of IIASA's Forest Sector Project and other units participated. A list of participants is given in Appendix B. Unlike other network meetings, the meeting was held in Sopron, Hungary, because accommodations were unexpectedly scarce near Vienna. The participants were appreciative of the gracious and generous hospitality of our hosts, the Hungarian Committee for Applied Systems Analysis. Formal presentations are only part of what happens at any coordinating meeting. Questions and comments during the meeting, conversations at breaks and meals, distribution of papers by those that could not attend, and separate meetings on special topics all contributed to accomplishments. This report attempts to summarize the meeting as a whole

Loss of cation-independent mannose 6-phosphate receptor expression promotes the accumulation of lysobisphosphatidic acid in multilamellar bodies

SUMMARYA number of recent studies have highlighted the importance of lipid domains within endocytic organelles in the sorting and movement of integral membrane proteins. In particular, considerable attention has become focussed upon the role of the unusual phospholipid lysobisphosphatidic acid (LBPA). This lipid appears to be directly involved in the trafficking of cholesterol and glycosphingolipids, and accumulates in a number of lysosomal storage disorders. Antibody-mediated disruption of LBPA function also leads to mis-sorting of cation-independent mannose 6-phosphate receptors. We now report that the converse is also true, and that spontaneous loss of cation-independent mannose 6- phosphate receptors from a rat fibroblast cell line led to the formation of aberrant late endocytic structures enriched in LBPA. Accumulation of LBPA was directly dependent upon the loss of the receptors, and could be reversed by expression of bovine cation-independent mannose 6- phosphate receptors in the mutant cell line.Ultrastructural analysis indicated that the abnormalorganelles were electron-dense, had a multi-lamellar structure, accumulated endocytosed probes, and were distinct from dense-core lysosomes present within the same cells. The late endocytic structures present at steady state within any particular cell likely reflect the balance of membrane traffic through the endocytic pathway of that cell, and the rate of maturation of individual endocytic organelles. Moreover, there is considerable evidence which suggests that cargo receptors also play a direct mechanistic role in membrane trafficking events. Therefore, loss of such a protein may disturb the overall equilibrium of the pathway, and hence cause the accumulation of aberrant organelles. We propose that this mechanism underlies the phenotype of the mutant cell line, and that the formation of inclusion bodies in many lysosomal storage diseases is also due to an imbalance in membrane trafficking within the endocytic pathway

Excellence and efficiency in the delivery system at pre-university level

In the current educational climate in Malaysia, the very relevance of pre-university programmes of study is increasingly being questioned. As such, a concerted drive by pre-university educators towards excellence and efficacy is vital if such programmes are to resist the eventual slide into oblivion. This paper aims to take a critical look at the delivery system. It identifies and discusses some inherent weaknesses in the system and emphasizes the role of the educator in shaping an environment that is conducive to effective learning whilst taking cognizance of the numerous factors outside the orbit of the educator that can compromise the effectual delivery of subject matter. The paper also makes recommendations towards the attainment of excellence and efficacy in the delivery system, drawing conclusions from a survey conducted amongst past and present students as well as actual classroom experience. These recommendations are based on personal opinions and broad pedagogical philosophies rather than on actual teaching methodologies

Overexpression of a rat kinase-deficient phosphoinositide 3-kinase, Vps34p, inhibits cathepsin D maturation

Lipid kinases and their phosphorylated products are important regulators of many cellular processes, including intracellular membrane traffic. The best example of this is provided by the class III phosphoinositide 3-kinase (PI-3K), Vps34p, which is required for correct targeting of newly synthesized carboxy- peptidase Y to the yeast vacuole. A probable mammalian Vps34p orthologue has been previously identified, but its function in the trafficking of lysosomal enzymes has not been resolved. To investigate the possible role(s) of mammalian Vps34p in protein targeting to lysosomes, we have cloned the rat orthologue and overexpressed a kinase-deficient mutant in HeLa cells. Expression of the mutant protein inhibited both maturation of procathepsin D and basal secretion of the precursor. In contrast wortmannin,INTRODUCTIONThere is now abundant evidence to indicate that, in addition to their roles in cell signalling, lipid kinases and their phosphorylated products are important regulators of intracellular membrane traffic (reviewed in [1–3]). Perhaps the best example of this is provided by the class III phosphoinositide 3-kinase (PI-3K), Vps34p. This enzyme was first identified as a component of the yeast vacuolar protein sorting machinery, which, when inacti- vated, causes aberrant secretion of newly synthesized carboxy- peptidase Y (CPY) from the yeast Golgi [4,5]. In addition to its role in biosynthetic traffic, there is also considerable evidence to support a requirement for Vps34p in the endocytic pathway. For example, yeast expressing end12 (a mutant allele of VPS34) are defective in transferring endocytosed α-factor to the vacuole [6], and Vac1p\Vps19p, which is required for fusion of transport vesicles to yeast endosomes, binds the lipid product of Vps34p [7,8]. Vac1p is one member of a family of proteins that contain the FYVE domain [3], which is implicated in PtdIns(3)P binding [9,10]. Other yeast family members include Vps27p and the PtdIns(3)P 5-kinase Fab1p. Deletion of FAB1 causes abnormally large vacuoles containing fewer internal vesicles than normal to form, suggesting that the lipid product is required for correct sorting in the yeast multivesicular body [11]. Vps34p is the sole PI-3K in Saccharomyces cereisiae [5], which implies that this enzyme has multiple roles throughout the yeast endocytic system.In general, the membrane trafficking machinery appears highly conserved between all eukaryotes. Hence the involvement of a Vps34p orthologue in targeting newly synthesized lysosomal hydrolases has been postulated on the basis that PI-3K inhibitors,which also inhibited maturation, caused hypersecretion of the precursor. We propose that mammalian Vps34p plays a direct role in targeting lysosomal enzyme precursors to the endocytic pathway in an analogous fashion to its role in the fusion of early endocytic vesicles with endosomes. We further suggest that inhibition of a wortmannin-sensitive enzyme, other than mam- malian Vps34p, is responsible for the failure to recycle unoccupied mannose 6-phosphate receptors to the trans-Golgi network, and consequent hypersecretion of lysosomal enzyme precursors observed in the presence of this drug

Land Grant Application- Row, Webber (Baldwin)

Land grant application submitted to the Maine Land Office for Webber Row for service in the Revolutionary War.https://digitalmaine.com/revolutionary_war_me_land_office/1779/thumbnail.jp