Proteasome mutants, pre4-2 and ump1-2, suppress the essential function but not the mitochondrial RNase P function of the Saccharomyces cerevisiae gene RPM2.

The Saccharomyces cerevisiae nuclear gene RPM2 encodes a component of the mitochondrial tRNA-processing enzyme RNase P. Cells grown on fermentable carbon sources do not require mitochondrial tRNA processing activity, but still require RPM2, indicating an additional function for the Rpm2 protein. RPM2-null cells arrest after 25 generations on fermentable media. Spontaneous mutations that suppress arrest occur with a frequency of approximately 9 x 10(-6). The resultant mutants do not grow on nonfermentable carbon sources. We identified two loci responsible for this suppression, which encode proteins that influence proteasome function or assembly. PRE4 is an essential gene encoding the beta-7 subunit of the 20S proteasome core. A Val-to-Phe substitution within a highly conserved region of Pre4p that disrupts proteasome function suppresses the growth arrest of RPM2-null cells on fermentable media. The other locus, UMP1, encodes a chaperone involved in 20S proteasome assembly. A nonsense mutation in UMP1 also disrupts proteasome function and suppresses Deltarpm2 growth arrest. In an RPM2 wild-type background, pre4-2 and ump1-2 strains fail to grow at restrictive temperatures on nonfermentable carbon sources. These data link proteasome activity with Rpm2p and mitochondrial function.

Cloned beta 1,4 N-acetylgalactosaminyltransferase synthesizes GA2 as well as gangliosides GM2 and GD2. GM3 synthesis has priority over GA2 synthesis for utilization of lactosylceramide substrate in vivo.

Earlier studies reached conflicting conclusions as to the ability of the beta 1,4 N-acetylgalactosaminyltransferase (GalNAc-T) that synthesizes gangliosides GM2 and GD2 to also produce gangliotriosylceramide (GA2). We constructed an experimental system in which to address this question. Wild type Chinese hamster ovary (CHO) cells contain ganglioside GM3 as the most complex glycosphingolipid (GSL), whereas the CHO glycosylation mutant Lec2, which is deficient in sialylation, accumulates lactosylceramide with little GM3 being produced. We transfected both cell types with a plasmid containing a cloned GalNAc-T. Whereas transfected CHO cells produced GM2 as the major complex GSL, the major product in transfected Lec2 cells was GA2. Both types of transfected cells but not the untransfected cells expressed the transfected gene and contained high levels of enzyme activity for synthesizing both GM2 and GA2 in vitro. In summary, these results indicate that this enzyme can in fact synthesize GA2 as well as GM2 and GD2. In addition, these findings suggest that in CHO cells the synthesis of GM3 in vivo has priority over GA2 synthesis for utilization of the substrate lactosylceramide, resulting in little GA2 being produced even though GalNAc-T is present and active. Thus, competition for substrate between glycosylation pathways may have profound effects on the GSL pattern of cells.

Transport of newly synthesized glucosylceramide to the plasma membrane by a non-Golgi pathway.

High-gradient magnetic affinity chromatography (HIMAC) has been used to obtain highly enriched plasma membranes, free of intracellular membrane contaminants, from cultured Chinese hamster ovary (CHO) cells in yields of > or = 80%. Using this procedure we have characterized the transport of glucosylceramide (GlcCer) and the ganglioside GM3 to the plasma membrane. Newly synthesized GlcCer reaches the plasma membrane in 7.2 min, whereas GM3 requires 21.5 min to reach the plasma membrane. Brefeldin A prevents transport of newly synthesized GM3 and sphingomyelin to the plasma membrane but has no effect on the transport of GlcCer. Similarly, incubation of CHO cells at 15 degrees C blocks transport of GM3 and sphingomyelin to the plasma membrane but has no effect on GlcCer movement. We propose that carrier-mediated transport accounts for a major fraction of the plasma membrane GlcCer. Pulse-chase studies with either [3H]glucose or [3H]palmitate indicate that newly synthesized GlcCer which has reached the plasma membrane is not utilized for the synthesis of GM3 but is instead rapidly either degraded or converted into an as yet unidentified product. Our results indicate that in addition to serving as a precursor for higher glycosylation in the Golgi, a major fraction of newly synthesized GlcCer is rapidly transported to the plasma membrane by a non-Golgi pathway and then rapidly turned over.

Determination of plasma membrane lipid mass and composition in cultured Chinese hamster ovary cells using high gradient magnetic affinity chromatography.

We have utilized wheat germ agglutinin conjugated to iron/dextran particles in conjunction with high gradient magnetic affinity chromatography (HIMAC) to prepare plasma membranes from cultured cells. Membrane-impermeable succinimidyl esters inactivate alkaline phosphodiesterase 1 (APDE-1) and were used to establish the proportion of APDE-1 expressed at the cell surface. The yield of inhibitable APDE-1 provides an accurate indication of plasma membrane yield, which was > 90% for Chinese hamster ovary (CHO) cells. Plasma membranes prepared by HIMAC contained < 5-13% of endoplasmic reticulum, Golgi, mitochondria, lysosomes, or endosomes. Pulse-chase experiments performed with the alpha 5 beta 1 integrin receptor confirmed the high yield of plasma membrane and demonstrated the utility of this procedure for examining trafficking of proteins to and from the plasma membrane. We determined the lipid content of plasma membranes prepared by HIMAC. CHO plasma membranes contain 49% of total cellular phospholipid, 69% of sphingomyelin, and 64% of cholesterol. Phosphatidylserine was the only glycerophospholipid highly enriched (71%) in the retained fraction. The glycosphingolipids lactosylceramide and ganglioside GM3 were enriched in the plasma membrane fraction to the same extent as sphingomyelin. The major fraction of the glycosphingolipid precursors glucosylceramide and ceramide was localized to intracellular membranes. These findings indicate that the plasma membrane of CHO cells contains approximately half of the total cellular phospholipids and an even higher percentage of sphingomyelin and cholesterol. The high efficiency and rapidity of this isolation procedure should aid the analysis of plasma membrane components significantly.

Endogenous glycosphingolipids move to the cell surface at a rate consistent with bulk flow estimates.

The bulk flow model of intracellular trafficking predicts that forward transport from the ER through the Golgi to the plasma membrane proceeds by default without a special signal being required (Wieland, F.T., Gleason, M. L., Serafini, T. A., and Rothman, J. E. (1987) Cell 50, 289-300). We tested a crucial prediction of this model, which is that the endogenous lipid components of the transport vesicles would reach the plasma membrane at the rapid rate of bulk flow. The rate at which endogenous glycosphingolipids moved from the ER through the Golgi to the plasma membrane was determined in Chinese hamster ovary cells using metabolic labeling with tritiated palmitate and oxidation of cell surface ganglioside NeuAc alpha 2----3Gal beta 1----4Glc beta 1----4Cer (GM3) with periodate. Whereas radioactive precursor became incorporated into ceramide and glucosyl ceramide without a detectable lag, synthesis of labeled lactosyl ceramide and ganglioside GM3 did not begin until 5-6 min and 11-12 min, respectively, after addition of labeled precursor. Labeled GM3 reached the plasma membrane 5-6 min following its synthesis. Overall, approximately 18 min transpired from the time that the ceramide precursor was synthesized in the ER until labeled GM3 reached the plasma membrane. These results indicate that lipid transport vesicles move rapidly to the plasma membrane at a rate consistent with bulk flow estimates.

Use of brefeldin A to define sites of glycosphingolipid synthesis: GA2/GM2/GD2 synthase is trans to the brefeldin A block.

Brefeldin A (BFA) induces the rapid redistribution of the Golgi complex into the endoplasmic reticulum (ER), causing the glycoproteins that are retained in the ER to be processed by Golgi enzymes. We have examined the effects of BFA on the synthesis of glycosphingolipids (GSL) to map the intracellular sites of GSL synthesis. In several cultured cell types, BFA inhibited the synthesis of the neutral GSL gangliotriaosylceramide (GA2) and monosialoganglioside GM2 and disialoganglioside GD2, where GD2 is GalNAc(beta 1----4)- [NeuAc(alpha 2----8)NeuAc(alpha 2----3)]Gal(beta 1----4)GlcCer, GM2 lacks the NeuAc(alpha 2----8) unit, and GA2 lacks both NeuAc(alpha 2----8) and NeuAc(alpha 2----3) units. The observed decrease in labeling of GA2, GM2, and GD2 in the presence of BFA was not due either to enhanced degradation of these glycolipids or to shedding of these glycolipids from the cells. In rat liver all three of these glycolipids have been shown by others to be synthesized by the same enzyme, GA2/GM2/GD2 synthase, which catalyzes the addition of N-acetylgalactosamine to lactosylceramide (Lac-Cer), GM3 [NeuAc(alpha 2----3)Gal(beta 1----4)GlcCer], and GD3 [NeuAc(alpha 2----8)NeuAc-(alpha 2----3)Gal(beta 1----4)GlcCer], respectively. Studies with a fluorescent glycolipid analog indicated that BFA redistributed the trans-Golgi stacks into a reticular pattern characteristic of the ER. These studies localize GA2/GM2/GD2 synthase, a key enzyme involved in the synthesis of complex gangliosides, to a compartment late in the intracellular trafficking pathway, which remains functionally distinct from the ER in the presence of BFA.

Identification of the gene for fly non-muscle myosin heavy chain: Drosophila myosin heavy chains are encoded by a gene family.

In contrast to vertebrate species Drosophila has a single myosin heavy chain gene that apparently encodes all sarcomeric heavy chain polypeptides. Flies also contain a cytoplasmic myosin heavy chain polypeptide that by immunological and peptide mapping criteria is clearly different from the major thoracic muscle isoform. Here, we identify the gene that encodes this cytoplasmic isoform and demonstrate that it is distinct from the muscle myosin heavy chain gene. Thus, fly myosin heavy chains are the products of a gene family. Our data suggest that the contractile function required to power myosin based movement in non-muscle cells requires myosin diversity beyond that available in a single heavy chain gene. In addition, we show, that accumulation of cytoplasmic myosin transcripts is regulated in a developmental stage specific fashion, consistent with a key role for this protein in the movements of early embryogenesis.