Linda J. Wuestehube

Reconstitution of transport from endoplasmic reticulum to Golgi complex using endoplasmic reticulum-enriched membrane fraction from yeast

Publisher Summary Protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus may be monitored in vitro with gently lysed semiintact cells or with crude homogenates fractionated by differential centrifugation. A complete resolution of this reaction requires the purification of donor ER and acceptor Golgi membranes. Toward this end there is a development of an in vitro assay that utilizes an ER-enriched membrane fraction to reconstitute ER-to-Golgi transport. This new assay allows a detailed analysis of the specific contributions of both membrane and soluble components to the transport process not possible in semiintact cells and crude homogenates. The development of an enriched ER in vitro assay represents a methodological advance in defining the molecular requirements for ER-to- Golgi transport. The enriched ER transport assay offers more flexibility than assays using semiintact cells in that membrane fractions from different sources can readily be mixed. The development of the enriched ER transport assay represents an advance toward the reconstitution of ER-to-Golgi transport in a fully resolved system. The use of this assay in analyzing the biochemical requirements of transport in vitro will aid in defining the molecular details of transport in vivo.

epsilon-COP is a structural component of coatomer that functions to stabilize alpha-COP.

We isolated a novel yeast α‐COP mutant, ret1‐3, in which α‐COP is degraded after cells are shifted to a restrictive temperature. ret1‐3 cells cease growth at 28°C and accumulate the ER precursor of carboxypeptidase Y (p1 CPY). In a screen for high copy suppressors of these defects, we isolated the previously unidentified yeast ϵ‐COP gene. ϵ‐COP (Sec28p) overproduction suppresses the defects of ret1‐3 cells up to 34°C, through stabilizing levels of α‐COP. Surprisingly, cells lacking ϵ‐COP (sec28Δ) grow well up to 34°C and display normal trafficking of carboxypeptidase Y and KKXX‐tagged proteins at a permissive temperature. ϵ‐COP is thus non‐essential for yeast cell growth, but sec28Δ cells are thermosensitive. In sec28Δ cells shifted to 37°C, wild‐type α‐COP (Ret1p) levels diminish rapidly and cells accumulate p1 CPY; these defects can be suppressed by α‐COP overproduction. Mutant coatomer from sec28Δ cells behaves as an unusually large protein complex in gel filtration experiments. The sec28Δ mutation displays allele‐specific synthetic‐lethal interactions with α‐COP mutations: sec28Δ ret1‐3 double mutants are unviable at all temperatures, whereas sec28Δ ret1‐1 double mutants grow well up to 30°C. Our results suggest that a function of ϵ‐COP is to stabilize α‐COP and the coatomer complex.

[5] F-actin affinity chromatography of detergent-solubilized plasma membranes: Purification and initial characterization of ponticulin from Dictyostelium discoideum

Publisher Summary This chapter describes the characteristics of ponticulin and describes the procedure of isolating it from detergent-solubilized Dictyostelium discoideum plasma membranes by elution with high salt from F-actin affinity columns followed by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Ponticulin purified by these methods has been used to obtain the amino acid composition and amino-terminal sequence data. The details for the preparation of 5 ml packed F-actin beads are presented. Ponticulin is a developmentally regulated protein that may be important for increased actin association with the plasma membrane during aggregation. The amount of ponticulin in the plasma membrane increases two- to threefold when D. discoideum amebas are forming aggregation streams, suggesting that this protein may play a role in the enhanced cell motility and/or cell-cell adhesion characteristic of this developmental stage. A relatively abundant protein in the plasma membrane, ponticulin constitutes about 1% of the total membrane protein, and appears to span the membrane. Ponticulin eluted from F-actin columns with high salt binds again to F-actin columns after the salt concentration is lowered to physiological levels by dialysis against 1% OG, CB. Furthermore, salt-eluted ponticulin subjected to SDS-polyacrylamide gel electrophoresis and electrotransfer to a nitrocellulose membrane directly and specifically binds 125 I-labeled F-actin.

Genes and proteins required for vesicular transport from the endoplasmic reticulum

Transport of proteins between compartments of the secretory pathway is mediated by small vesicles which bud from a donor compartment and fuse with a target compartment (Palade 1975). Transport vesicles that mediate ER to Golgi transport have been identified in vivo in mammalian cells (Jamieson & Palade 1967) and yeast cells (Novick et al. 1981; Kaiser & Schekman 1990). Membrane fractions with properties suggestive of an intermediate compartment between the ER and the Golgi have also been generated in vitro in mammalian (Paulik et al. 1988) and yeast lysates (Groesch et al. 1990). Ten sec mutations block protein transport from the ER to the Golgi apparatus at 37 ~ (Novick et al. 1980). By electron microscopy, a subset of these mutants, the class II mutants (secl7, secl8, and sec22), accumulate 50 nm vesicles and enlarged ER structures at 37 ~ A second subset, the class I mutants (secl2, secl3, secl6, sec23), accumulate ER structures at 37 ~ and block the accumulation of vesicles when in double-mutant combination with any of the class II sec genes (Kaiser & Schekman 1990). Thus class I Sec proteins are thought to be required for transport vesicle budding and class II Sec proteins, for vesicle fusion (Fig. 1). Yeast carrying a mutation in the ras-like gene YPT1 (Gallwitz 1983) accumulate enlarged ER and Golgi structures at the nonpermissive temperatures (Segev et al. 1988; Schmitt et al. 1988). Vesicle mediated transport in vitro