Mechanism of residence of cytochrome b(5), a tail-anchored protein, in the endoplasmic reticulum

Endoplasmic reticulum (ER) proteins maintain their residency by static retention, dynamic retrieval, or a combination of the two. Tail-anchored proteins that contain a cytosolic domain associated with the lipid bilayer via a hydrophobic stretch close to the COOH terminus are sorted within the secretory pathway by largely unknown mechanisms. Here, we have investigated the mode of insertion in the bilayer and the intracellular trafficking of cytochrome b(5) (b[5]), taken as a model for ER-resident tail-anchored proteins. We first demonstrated that b(5) can acquire a transmembrane topology posttranslationally, and then used two tagged versions of b(5), N-glyc and O-glyc b(5), containing potential N- and O-glycosylation sites, respectively, at the COOH-terminal lumenal extremity, to discriminate between retention and retrieval mechanisms. Whereas the N-linked oligosaccharide provided no evidence for retrieval from a downstream compartment, a more stringent assay based on carbohydrate acquisition by O-glyc b(5) showed that b(5) gains access to enzymes catalyzing the first steps of O-glycosylation. These results suggest that b(5) slowly recycles between the ER and the cis-Golgi complex and that dynamic retrieval as well as retention are involved in sorting of tail-anchored proteins.     

Effects of inositol 1,4,5-trisphosphate on calcium release from the endoplasmic reticulum and Golgi apparatus in mouse mammary epithelial cells: a comparison during pregnancy and lactation

It has been established that inositol 1,4,5-trisphosphate(IP3) is responsible for the mobilization of calcium(Ca2+) from intracellular locations in a wide variety of tissues, and that this response triggers the stimulation of several hormones and neurotransmitters. However, these phenomena have yet to be examined in the mammary epithelium. Ca2+ uptake from the medium into the endoplasmic reticulum(ER) and Golgi apparatus in vitro in both pregnant and lactating mouse mammary epithelial cells was studied and a strong Ca2+ release from these organelles into the medium with the use of IP3 was shown. The Ca2+ uptake and its release due to IP3 was also usually greater during pregnancy than lactation.     

Asymmetric requirements for a Rab GTPase and SNARE proteins in fusion of COPII vesicles with acceptor membranes

Soluble NSF attachment protein receptor (SNARE) proteins are essential for membrane fusion in transport between the yeast ER and Golgi compartments. Subcellular fractionation experiments demonstrate that the ER/Golgi SNAREs Bos1p, Sec22p, Bet1p, Sed5p, and the Rab protein, Ypt1p, are distributed similarly but localize primarily with Golgi membranes. All of these SNARE proteins are efficiently packaged into COPII vesicles and suggest a dynamic cycling of SNARE machinery between ER and Golgi compartments. Ypt1p is not efficiently packaged into vesicles under these conditions. To determine in which membranes protein function is required, temperature-sensitive alleles of BOS1, BET1, SED5, SLY1, and YPT1 that prevent ER/Golgi transport in vitro at restrictive temperatures were used to selectively inactivate these gene products on vesicles or on Golgi membranes. Vesicles bearing mutations in Bet1p or Bos1p inhibit fusion with wild-type acceptor membranes, but acceptor membranes containing these mutations are fully functional. In contrast, vesicles bearing mutations in Sed5p, Sly1p, or Ypt1p are functional, whereas acceptor membranes containing these mutations block fusion. Thus, this set of SNARE proteins is symmetrically distributed between vesicle and acceptor compartments, but they function asymmetrically such that Bet1p and Bos1p are required on vesicles and Sed5p activity is required on acceptor membranes. We propose the asymmetry in SNARE protein function is maintained by an asymmetric distribution and requirement for the Ypt1p GTPase in this fusion event. When a transmembrane-anchored form of Ypt1p is used to restrict this GTPase to the acceptor compartment, vesicles depleted of Ypt1p remain competent for fusion.     

A role for actin, Cdc1p, and Myo2p in the inheritance of late Golgi elements in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Golgi elements are present in the bud very early in the cell cycle. We have analyzed this Golgi inheritance process using fluorescence microscopy and genetics. In rapidly growing cells, late Golgi elements show an actin-dependent concentration at sites of polarized growth. Late Golgi elements are apparently transported into the bud along actin cables and are also retained in the bud by a mechanism that may involve actin. A visual screen for mutants defective in the inheritance of late Golgi elements yielded multiple alleles of CDC1. Mutations in CDC1 severely depolarize the actin cytoskeleton, and these mutations prevent late Golgi elements from being retained in the bud. The efficient localization of late Golgi elements to the bud requires the type V myosin Myo2p, further suggesting that actin plays a role in Golgi inheritance. Surprisingly, early and late Golgi elements are inherited by different pathways, with early Golgi elements localizing to the bud in a Cdc1p- and Myo2p-independent manner. We propose that early Golgi elements arise from ER membranes that are present in the bud. These two pathways of Golgi inheritance in S. cerevisiae resemble Golgi inheritance pathways in vertebrate cells.     

Golgi clusters and vesicles mediate mitotic inheritance independently of the endoplasmic reticulum

We have examined the fate of Golgi membranes during mitotic inheritance in animal cells using four-dimensional fluorescence microscopy, serial section reconstruction of electron micrographs, and peroxidase cytochemistry to track the fate of a Golgi enzyme fused to horseradish peroxidase. All three approaches show that partitioning of Golgi membranes is mediated by Golgi clusters that persist throughout mitosis, together with shed vesicles that are often found associated with spindle microtubules. We have been unable to find evidence that Golgi membranes fuse during the later phases of mitosis with the endoplasmic reticulum (ER) as a strategy for Golgi partitioning (Zaal, K.J., C.L. Smith, R.S. Polishchuk, N. Altan, N.B. Cole, J. Ellenberg, K. Hirschberg, J.F. Presley, T.H. Roberts, E. Siggia, et al. 1999. Cell. 99:589-601) and suggest that these results, in part, are the consequence of slow or abortive folding of GFP-Golgi chimeras in the ER. Furthermore, we show that accurate partitioning is accomplished early in mitosis, by a process of cytoplasmic redistribution of Golgi fragments and vesicles yielding a balance of Golgi membranes on either side of the metaphase plate before cell division.     

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.     

Discovery of the molecular machinery CERT for endoplasmic reticulum-to-Golgi trafficking of ceramide

Synthesis and sorting of lipids are essential events for membrane biogenesis and its homeostasis. Ceramide is synthesised at the endoplasmic reticulum (ER), and translocated to the Golgi compartment for conversion to sphingomyelin (SM). We have recently identified a key factor (named CERT) for ceramide trafficking. In this short review, I summarise recent advances in molecular mechanisms of intracellular transport of ceramide, focusing on our genetic and biochemical approaches to this issue.     

Selective Accumulation of the Endoplasmic Reticulum–Golgi Intermediate Compartment Induced by the Antitumor Drug KRN5500

KRN5500 is a semisynthetic spicamycin analogue consisting of a seven-carbon amino sugar linked to a C₁₄ unsaturated fatty acid through glycine and to the amino group of adenine. The drug inhibits cell growth potently and has antitumor activity in in vivo models. The mechanism of the antiproliferative effect of KRN5500 remains to be elucidated. We have found that acute exposure of drug-sensitive HT-29 colon adenocarcinoma cells to the drug results initially in swelling of the Golgi apparatus. Continuous exposure to the drug resulted in the emergence of a resistant population of cells characterized by numerous intracellular vacuoles. These KRN5500-resistant tumor cells exhibited increased staining with the Golgi stain NBD C₆–ceramide and the ER–Golgi fluorescent dye BODIPY–brefeldin A, which, unlike the parental drug-sensitive cells, was dispersed throughout the cytoplasm. Marker enzymes associated with the ER (glucose 6-phosphatase) and cis-Golgi (GalNAc transferase) were elevated >2-fold and nearly 4-fold, respectively, in drug-resistant cell lines while the trans-Golgi marker enzyme, galactosyltransferase, was not. The additional findings that the KRN5500-resistant cells have a >2-fold elevation in ERGIC-53, a cis-Golgi marker protein of the ER–Golgi intermediate compartment (ERGIC), as well as increased 58K, a 58-kDa microtubule-binding protein with formiminotransferase cyclodeaminase activity, and tubulin indicate that the cellular secretory pathway is a primary determinant of sensitivity to KRN5500, as resistance to this agent corresponds with accumulation of several components relatable to ER and cis-Golgi function. Further support for this conclusion is provided by studies which demonstrate that KRN5500 alters the distribution of newly synthesized carcinoembryonic antigen within the secretory pathway, including arrest of this N-glycosylated protein in the Golgi of LS-174T colon carcinoma cells.     

Comparison of Golgi apparatus and endoplasmic reticulum proteins from livers of juvenile and aged rats using a novel technique for separation and enrichment of organelles.

The broad dynamic range of protein abundances, which can vary from about 10(6) for cells to 10(10) for tissues in complex proteomes, continues to challenge proteomics research. Proteome analysis, in particular organelle proteomics, using current approaches, requires extensive fractionation, separation, and enrichment. Over the years, organelle separation was achieved through the use of differential and density-gradient ultracentrifugation. However, the traditional fixed-volume process is a time-consuming and labor-intensive method, especially with large quantities of sample. Here, we present a novel tool for subcellular fractionation of biologically complex mixtures: continuous-flow ultracentrifugation of tissue homogenates to obtain both organelle separation and extensive organelle enrichment at the same time. In this study, rat liver tissues from two different age groups (3-8 wk and greater than 1 y old) were homogenized by blending. After removing nuclei, the resulting homogenates were further fractionated at the subcellular level by the use of sucrose gradient continuous-flow ultracentrifugation. Each organelle's enriched fractions were identified by Western blot analysis. To study the possible effects of aging on the endoplasmic reticulum and Golgi apparatus, we compared the organelle protein profiles of the two groups of rat liver tissues using two-dimensional gel electrophoresis, digitized imaging of two-dimensional gel electrophoresis, and mass spectrometry. Significant differences in the protein profiles of both organelles were observed between the two groups of rat tissues. The technique described here for fractionation and enrichment of organelles demonstrated a useful tool for proteomics research, including identification of low-abundance proteins and post-translational modifications.     

Recycling of Golgi-resident Glycosyltransferases through the ER Reveals a Novel Pathway and Provides an Explanation for Nocodazole-induced Golgi Scattering

During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway. To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11–amino acid epitope, VSV-G (VSV), and the trans/TGN enzyme β1,4-galactosyltransferase (GalT) fused to GFP. After nocodazole addition, time-lapse microscopy of GalNAc-T2–GFP and GalT–GFP revealed that scattered Golgi elements appeared abruptly and that no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fluorescence intensity for tens of minutes. During the entire process of dispersal, immunogold labeling for GalNAc-T2–VSV and GalT showed that these were continuously concentrated over stacked Golgi cisternae and tubulovesicular Golgi structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites. In fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident proteins with each other through what appeared to be an ER intermediate. That Golgi enzymes cycle through the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1p^dn) blocking ER export. Sar1p^dn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumulation of GalNAc-T2–VSV in the ER. Few to no peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1p^dn. In conclusion, we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced Golgi scattering observed in interphase cells.