Distinct stages in the recognition, sorting, and packaging of proTGFα into COPII-coated transport vesicles.

In addition to its role in forming vesicles from the endoplasmic reticulum (ER), the coat protein complex II (COPII) is also responsible for selecting specific cargo proteins to be packaged into COPII transport vesicles. Comparison of COPII vesicle formation in mammalian systems and in yeast suggested that the former uses more elaborate mechanisms for cargo recognition, presumably to cope with a significantly expanded repertoire of cargo that transits the secretory pathway. Using proTGFα, the transmembrane precursor of transforming growth factor α (TGFα), as a model cargo protein, we demonstrate in cell-free assays that at least one auxiliary cytosolic factor is specifically required for the efficient packaging of proTGFα into COPII vesicles. Using a knockout HeLa cell line generated by CRISPR/Cas9, we provide functional evidence showing that a transmembrane protein, Cornichon-1 (CNIH), acts as a cargo receptor of proTGFα. We show that both CNIH and the auxiliary cytosolic factor(s) are required for efficient recruitment of proTGFα to the COPII coat in vitro. Moreover, we provide evidence that the recruitment of cargo protein by the COPII coat precedes and may be distinct from subsequent cargo packaging into COPII vesicles

Invertase signal and mature sequence substitutions that delay intercompartmental transport of active enzyme

The role of structural signals in intercompartmental transport has been addressed by the isolation of yeast invertase (SUC2) mutations that cause intracellular accumulation of active enzyme. Two mutations that delay transport of core-glycosylated invertase, but not acid phosphatase, have been mapped in the 5' coding region of SUC2. Both mutations reduce specifically the transport of invertase to a compartment, presumably in the Golgi body, where outer chain carbohydrate is added. Subsequent transport to the cell surface is not similarly delayed. One mutation (SUC2-s1) converts an ala codon to val at position -1 in the signal peptide; the other (SUC2-s2) changes a thr to an ile at position +64 in the mature protein. Mutation s1 results in about a 50-fold reduced rate of invertase transport to the Golgi body which is attributable to defective signal peptide cleavage. While peptide cleavage normally occurs at an ala-ser bond, the s1 mutant form is processed slowly at the adjacent ser-met position giving rise to mature invertase with an N-terminal met residue. s2 mutant invertase is transported about sevenfold more slowly than normal, with no delay in signal peptide cleavage, and no detectable abnormal physical property of the enzyme. This substitution may interfere with the interaction of invertase and a receptor that facilitates transport to the Golgi body

Reflection on the enduring impact of Prof. Luis Leloir’s discoveries that led to his 1970 Nobel Prize in Chemistry

Reflexiones del Premio Nobel Randy Schekman acerca del impacto de los descubrimientos de Luis Leloir que llevaron a recibir el Premio Nobel en 1970. Texto redactado en ocasión del 50º aniversario del otorgamiento del Premio Nobel de Química a Luis F. Leloir.originalFil: Schekman, Randy.1 página en papelLFL-CD-OTROS. Escritos de OtrosUnidad documental simpl

Measurement of aileron hinge moments and the effect of aileron trailing edge thickness

A dissertation submitted to the Faculty of Engineering and the Built Environment, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Engineering. Johannesburg, July-2015The objective of this research was twofold. The first objective was for the design and testing of a compact force measurement device, capable of fitting inside the wind tunnel model and measuring aileron hinge moments. Using the hinge moment balance, the second objective was to test the effect of varying the trailing edge thickness of the aileron. A plain type aileron, with a 20.6% chord and 40% span, was attached to a NACA 0012 wing. Four aileron test pieces with trailing edge thicknesses from 0.39%c to 1.22%c were used. A external balance capable of measuring the wing rolling and yawing moments was used in conjunction with the hinge moment balance. Validation of the results and performance of the hinge moment balance was done by comparisons to the data obtained from previous research and investigations using flow visualization. The results indicated that the system performed in a manner that was expected and predictable. The data from the hinge moment balance had an uncertainty of 4.2% which was deemed satisfactory for the purposes of this research. For the majority of the test cases the effects of the varied trailing edge thickness was found to be negligible. Small differences were noted at high angles of attack, above 15°, and high aileron deflections, above 20°. As these conditions were seen to be outside the typical flight envelope of general aviation aircraft, these small differences were seen as inconsequential. It was concluded that the use of an aileron with a thicker trailing edge would not have a negative effect on the performance of the aileron while allowing for less restrictions on the manufacture of the aileron itself

Reconstitution of Retrograde Transport from the Golgi to the ER In Vitro

Retrograde transport from the Golgi to the ER is an essential process. Resident ER proteins that escape the ER and proteins that cycle between the Golgi and the ER must be retrieved. The interdependence of anterograde and retrograde vesicle trafficking makes the dissection of both processes difficult in vivo. We have developed an in vitro system that measures the retrieval of a soluble reporter protein, the precursor of the yeast pheromone α-factor fused to a retrieval signal (HDEL) at its COOH terminus (Dean, N., and H.R.B Pelham. 1990. J. Cell Biol. 111:369–377). Retrieval depends on the HDEL sequence; the α-factor precursor, naturally lacking this sequence, is not retrieved. A full cycle of anterograde and retrograde transport requires a simple set of purified cytosolic proteins, including Sec18p, the Lma1p complex, Uso1p, coatomer, and Arf1p. Among the membrane-bound v-SNAP receptor (v-SNARE) proteins, Bos1p is required only for forward transport, Sec22p only for retrograde trafficking, and Bet1p is implicated in both avenues of transport. Putative retrograde carriers (COPI vesicles) generated from Golgi-enriched membranes contain v-SNAREs as well as Emp47p as cargo

A subset of yeast vacuolar protein sorting mutants is blocked in one branch of the exocytic pathway

Exocytic vesicles that accumulate in a temperature-sensitive sec6 mutant at a restrictive temperature can be separated into at least two populations with different buoyant densities and unique cargo molecules. Using a sec6 mutant background to isolate vesicles, we have found that vacuolar protein sorting mutants that block an endosome-mediated route to the vacuole, including vps1, pep12, vps4, and a temperature-sensitive clathrin mutant, missort cargo normally transported by dense exocytic vesicles, such as invertase, into light exocytic vesicles, whereas transport of cargo specific to the light exocytic vesicles appears unaffected. Immunoisolation experiments confirm that missorting, rather than a changed property of the normally dense vesicles, is responsible for the altered density gradient fractionation profile. The vps41Δ and apl6Δ mutants, which block transport of only the subset of vacuolar proteins that bypasses endosomes, sort exocytic cargo normally. Furthermore, a vps10Δ sec6 mutant, which lacks the sorting receptor for carboxypeptidase Y (CPY), accumulates both invertase and CPY in dense vesicles. These results suggest that at least one branch of the yeast exocytic pathway transits through endosomes before reaching the cell surface. Consistent with this possibility, we show that immunoisolated clathrin-coated vesicles contain invertase