Evidence for Random Distribution and Translational Movement of Cytochrome b5 in Endoplasmic Reticulum

Most types of cells are known from their structure and overall form to possess a characteristic organization. In some instances this is evident in the non-random disposition of organelles and such system subunits as cisternae of the endoplasmic reticulum or the Golgi complex. In others it appears in the distribution and orientation of cytoplasmic fibrils. And in yet others the organization finds expression in the non-random distribution and orientation of microtubules, especially as found in highly anisometric cells and cell processes. The impression is unavoidable that in none of these cases is the organization achieved without the involvement of the cytoplasmic ground substance (CGS) or matrix. This impression is based on the fact that a matrix is present and that in all instances these formed structures, whether membranelimited or filamentous, are suspended in it. In some well-known instances, as in arrays of microtubules which make up axonemes and axostyles, the matrix resolves itself into bridges (and spokes) between the microtubules, bridges which are in some cases very regularly disposed and uniform in size (Mcintosh, 1973; Bloodgood and Miller, 1974; Warner and Satir, 1974).

Download Full-text

A mutant cytochrome b5 with a lengthened membrane anchor escapes from the endoplasmic reticulum and reaches the plasma membrane.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.93.9.4207 ◽

1996 ◽

Vol 93 (9) ◽

pp. 4207-4212 ◽

Cited By ~ 70

Author(s):

E. Pedrazzini ◽

A. Villa ◽

N. Borgese

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Cytochrome B5 ◽

Membrane Anchor

Download Full-text

The C-terminus of cytochrome b5 confers endoplasmic reticulum specificity by preventing spontaneous insertion into membranes

Biochemical Journal ◽

10.1042/bj20060990 ◽

2007 ◽

Vol 401 (3) ◽

pp. 701-709 ◽

Cited By ~ 13

Author(s):

Matthew P. A. Henderson ◽

Yeen Ting Hwang ◽

John M. Dyer ◽

Robert T. Mullen ◽

David W. Andrews

Keyword(s):

Endoplasmic Reticulum ◽

Subcellular Localization ◽

Lipid Bilayers ◽

Fusion Proteins ◽

Molecular Mechanisms ◽

Cytochrome B5 ◽

Terminal Sequence ◽

C Terminus

The molecular mechanisms that determine the correct subcellular localization of proteins targeted to membranes by tail-anchor sequences are poorly defined. Previously, we showed that two isoforms of the tung oil tree [Vernicia (Aleurites) fordii] tail-anchored Cb5 (cytochrome b5) target specifically to ER (endoplasmic reticulum) membranes both in vivo and in vitro [Hwang, Pelitire, Henderson, Andrews, Dyer and Mullen (2004) Plant Cell 16, 3002–3019]. In the present study, we examine the targeting of various tung Cb5 fusion proteins and truncation mutants to purified intracellular membranes in vitro in order to assess the importance of the charged CTS (C-terminal sequence) in targeting to specific membranes. Removal of the CTS from tung Cb5 proteins resulted in efficient binding to both ER and mitochondria. Results from organelle competition, liposome-binding and membrane proteolysis experiments demonstrated that removal of the CTS results in spontaneous insertion of tung Cb5 proteins into lipid bilayers. Our results indicate that the CTSs from plant Cb5 proteins provide ER specificity by preventing spontaneous insertion into incorrect subcellular membranes.

Download Full-text

The cytochrome b5 dependent C-5(6) sterol desaturase DES5A from the endoplasmic reticulum of Tetrahymena thermophila complements ergosterol biosynthesis mutants in Saccharomyces cerevisiae

Steroids ◽

10.1016/j.steroids.2012.08.015 ◽

2012 ◽

Vol 77 (13) ◽

pp. 1313-1320 ◽

Cited By ~ 14

Author(s):

Tomas J. Poklepovich ◽

Mauro A. Rinaldi ◽

Mariela L. Tomazic ◽

Nicolas O. Favale ◽

Aaron P. Turkewitz ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Tetrahymena Thermophila ◽

Cytochrome B5 ◽

Ergosterol Biosynthesis

Download Full-text

NUCLEAR MEMBRANES FROM MAMMALIAN LIVER

The Journal of Cell Biology ◽

10.1083/jcb.46.2.379 ◽

1970 ◽

Vol 46 (2) ◽

pp. 379-395 ◽

Cited By ~ 142

Author(s):

Werner W. Franke ◽

Barbara Deumling ◽

Baerbel Ermen ◽

Ernst-Dieter Jarasch ◽

Hans Kleinig

Keyword(s):

Endoplasmic Reticulum ◽

Nuclear Envelope ◽

Nuclear Membrane ◽

Cytochrome B5 ◽

Buoyant Density ◽

Nuclear Pore ◽

Nuclear Membranes ◽

Dna And Rna ◽

Mammalian Liver ◽

Order Of Magnitude

Nuclear membranes were isolated from rat and pig liver by sonication of highly purified nuclear fractions and subsequent removal of adhering nucleoproteins in a high salt medium. The fractions were examined in the electron microscope by both negative staining and thin sectioning techniques and were found to consist of nuclear envelope fragments of widely varying sizes. Nuclear pore complex constituents still could frequently be recognized. The chemical composition of the nuclear membrane fractions was determined and compared with those of microsomal fractions prepared in parallel. For total nuclei as well as for nuclear membranes and microsomes, various enzyme activities were studied. The results indicate that a similarity exists between both fractions of cytomembranes, nuclear envelope, and endoplasmic reticulum, with respect to their RNA:protein ratio and their content of polar and nonpolar lipids. Both membranous fractions had many proteins in common including some membrane-bound enzymes. Activities in Mg-ATPase and the two examined cytochrome reductases were of the same order of magnitude. The content of cytochrome b5 as well as of P-450 was markedly lower in the nuclear membranes. The nuclear membranes were found to have a higher buoyant density and to be richer in protein. The glucose-6-phosphatase and Na-K-ATPase activities in the nuclear membrane fraction were very low. In the gel electrophoresis, in addition to many common protein bands, some characteristic ones for either microsomal or nuclear membranous material were detected. Significant small amounts of DNA and RNA were found to remain closely associated with the nuclear envelope fragments. Our findings indicate that nuclear and endoplasmic reticulum membranes which are known to be in morphological continuity have, besides a far-reaching similarity, some characteristic differences.

Download Full-text

Redox constituents in milk fat globule membranes and rough endoplasmic reticulum from lactating mammary gland.

The Journal of Cell Biology ◽

10.1083/jcb.73.1.223 ◽

1977 ◽

Vol 73 (1) ◽

pp. 223-241 ◽

Cited By ~ 72

Author(s):

E D Jarasch ◽

G Bruder ◽

T W Keenan ◽

W W Franke

Keyword(s):

Endoplasmic Reticulum ◽

Mammary Gland ◽

Low Temperatures ◽

Milk Fat ◽

Cytochrome B5 ◽

Alpha Band ◽

Lactating Mammary Gland ◽

Milk Fat Globule ◽

Fat Globule ◽

Milk Fat Globule Membranes

Milk fat globule membranes (MFGM) and rough endoplasmic reticulum (RER) membranes were isolated from milk and lactating mammary gland from the cow and were characterized by biochemical and electron microscope methods in terms of gross composition (proteins, phospholipids, neutral lipids, cholesterol, RNA, and DNA) and purity. Both fractions contained significant amounts of a b-type cytochrome with several properties similar to those of cytochrome b5 from liver, as well as a rotenone-insensitive NADH- and NADPH-cytochrome c reductase. The b-type cytochrome content in the apical plasma membrane-derived MFGM was of the same order of magnitude as it was in RER membranes. It was characterized by a high resistance to extraction by low- and high-salt concentrations and nonionic detergents. MFGM contained much more flavin and much higher activities of xanthine oxidase than the RER membranes. The same redox components were found in MFGM and mammary RER from women, rats, mice, and goats, but in absolute contents great differences between the species were noted. The cytochromes described here differed from liver cytochrome b5 in some spectral properties. The alpha-band of the reduced hepatic cytochrome b5 is asymmetric with a maximum at 555 nm that is split into two distinct peaks at low temperatures. The alpha-band of the b-type cytochromes from MFGM and mammary RER appears as one symmetrical peak at about 560 nm that is not split at low temperatures. When treated with cyanide, MFGM and mammary microsomes showed difference spectra of a reduced b-type cytochrome. Under the same conditions, liver microsomes gave a completely different spectrum. These findings demonstrate the presence of a b-type cytochrome and associated redox enzymes in MFGM, i.e., a derivative of the apical cell surface membrane that is regularly used for envelopment of the milk fat globule during secretion.

Download Full-text

Both the outer mitochondrial membrane and the microsomal forms of cytochrome b5 reductase contain covalently bound myristic acid. Quantitative analysis on the polyvinylidene difluoride-immobilized proteins

Biochemical Journal ◽

10.1042/bj2660341 ◽

1990 ◽

Vol 266 (2) ◽

pp. 341-347 ◽

Cited By ~ 11

Author(s):

N Borgese ◽

R Longhi

Keyword(s):

Fatty Acids ◽

Endoplasmic Reticulum ◽

Myristic Acid ◽

Cytochrome B5 ◽

Molar Ratio ◽

Mitochondrial Membranes ◽

Polyvinylidene Difluoride ◽

Cytochrome B5 Reductase ◽

Fatty Acylation ◽

Mitochondrial Fractions

NADH-cytochrome b5 reductase is known to be located on two distinct membranes, i.e. endoplasmic reticulum and outer mitochondrial membranes. The endoplasmic-reticulum-associated form of the enzyme contains myristic acid in an amide linkage to its N-terminal glycine [Ozols, Carr & Strittmatter (1984) J. Biol. Chem. 259, 13349-13354]. To investigate whether the dual subcellular localization of the reductase corresponds to a difference in fatty acylation, the enzyme was purified from well-characterized rat liver microsomal and mitochondrial fractions and analysed by a new quantitative analytical procedure. The purified reductases were run on SDS/polyacrylamide gels and blotted on to polyvinylidene difluoride membranes. The reductase-containing bands were treated with hydroxylamine, and amide-linked fatty acids were then detached by acid hydrolysis. The detached fatty acids were extracted, derivatized and analysed as phenylacyl esters by reverse-phase h.p.l.c., and the protein content of the samples was determined by amino acid analysis of the acid hydrolysates. Myristic acid was found in both the microsomal and mitochondrial reductases in a molar ratio of 1:1 with protein. These results demonstrate for the first time the presence of a myristylated protein on outer mitochondrial membranes, and show that the microsomal and mitochondrial reductases are also identical in their fatty acylation.

Download Full-text

ONTOGENETIC CHANGES OF PROTEINS OF ENDOPLASMIC RETICULUM

The Journal of Cell Biology ◽

10.1083/jcb.52.3.733 ◽

1972 ◽

Vol 52 (3) ◽

pp. 733-742 ◽

Cited By ~ 6

Author(s):

Owen Black ◽

Edward Bresnick

Keyword(s):

Amino Acids ◽

Endoplasmic Reticulum ◽

Aminolevulinic Acid ◽

Fetal Liver ◽

Cytochrome B5 ◽

Male Rats ◽

Liver Development ◽

Major Protein ◽

Ontogenetic Changes ◽

Cytochrome P 450

The proteins of the smooth and rough endoplasmic reticulum from fetal, immature, and adult male rats were compared after incorporation of two radioactively labeled precursors, 14C-labeled amino acids and δ-aminolevulinic acid-3H by means of gel electrophoresis. The labeling patterns indicated that protein components present in two major electrophoretic bands underwent significant synthesis in fetal tissue while three actively incorporating protein bands were noted in adult tissue. Although the uptake of the amino acids-14C decreased for the smooth and rough elements of the endoplasmic reticulum as a whole during liver development, the qualitative patterns were not significantly different in adult and fetal livers. The over-all incorporation (disintegrations per minute per milligram protein) of the heme precursor into the smooth and rough elements also did not change with development. However, a change was noted in the distributional electrophoretic patterns with development. The estimation of molecular weight (by disc electrophoresis) and the incorporation of the heme precursor suggested the similarity of the two major protein bands to cytochrome P-450 and cytochrome b5, components of the endoplasmic reticulum, thought to be involved in the mixed-function oxidase system. The evidence indicated that in fetal liver, at a time when the oxidase capability was low, the amino acid incorporation into these two protein groups was the same as in the adult. The incorporation of the heme moiety, however, was different, decreasing in the cytochrome b5 region and increasing in the cytochrome P-450 region during development. These results correlate with the increase in oxidase activity associated with liver development.

Download Full-text