ABSTRACT
Advances in mass spectrometry combined with accelerated progress in genome sequencing projects have facilitated the rapid identification of proteins by enzymatic digestion, mass analysis, and sequence database searching. Applications for this technology range from the surveillance of protein expression in cells, tissues, and whole organisms, to the identification of proteins and posttranslational modifications. Here we consider practical aspects of the application of mass spectrometry in cell biology and illustrate these with examples from our own laboratories.
Subject(s)
Mass Spectrometry/methods , Sequence Analysis, Protein/methods , Amino Acid Sequence , Databases, Factual , Glycoproteins/chemistry , Molecular Sequence Data , Peptide Fragments/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The p24 family of type I integral-membrane proteins, which are localised in the endoplasmic reticulum (ER), the intermediate compartment and the Golgi apparatus, are thought to function as receptors for cargo exit from the ER and in transport vesicle formation. Members of the p24 family have been found in a molecular complex and are enriched in COPI-coated vesicles, which are involved in membrane traffic between the ER and Golgi complex. Although expressed abundantly, simultaneous deletion of several family members does not appear to affect cell viability and protein secretion in yeast. In order to gain more insights into the physiological roles of different p24 proteins, we generated mice deficient in the expression of one family member, p23 (also called 24delta1, see for alternative nomenclature). In contrast to yeast genetics, in mice disruption of both p23 alleles resulted in early embryonic lethality. Inactivation of one allele led not only to reduced levels of p23 itself but also to reduced levels of other family members. The reduction in steady-state protein levels also induced structural changes in the Golgi apparatus, such as the formation of dilated saccules. The generation of mice deficient in p23 expression has revealed an essential and non-redundant role for p23 in the earliest stages of mammalian development. It has also provided genetic evidence for the participation of p24 family members in oligomeric complexes and indicates a structural role for these proteins in maintaining the integrity of the early secretory pathway.
Subject(s)
Coated Vesicles/metabolism , Embryonic and Fetal Development/physiology , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Membrane Proteins/physiology , Receptors, Cytoplasmic and Nuclear , Alleles , Animals , Biological Transport , Blotting, Northern , Blotting, Western , Coatomer Protein/metabolism , Embryonic and Fetal Development/genetics , Gene Targeting , Genes, Lethal , Genotype , Golgi Apparatus/ultrastructure , Macromolecular Substances , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Knockout , Multigene Family , Subcellular Fractions/chemistryABSTRACT
Six new members of the yeast p24 family have been identified and characterized. These six genes, named ERP1-ERP6 (for Emp24p- and Erv25p-related proteins) are not essential, but deletion of ERP1 or ERP2 causes defects in the transport of Gas1p, in the retention of BiP, and deletion of ERP1 results in the suppression of a temperature-sensitive mutation in SEC13 encoding a COPII vesicle coat protein. These phenotypes are similar to those caused by deletion of EMP24 or ERV25, two previously identified genes that encode related p24 proteins. Genetic and biochemical studies demonstrate that Erp1p and Erp2p function in a heteromeric complex with Emp24p and Erv25p.
Subject(s)
Carrier Proteins/metabolism , Fungal Proteins/metabolism , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins , Vesicular Transport Proteins , Yeasts/metabolism , Amino Acid Sequence , Base Sequence , Biological Transport , Carrier Proteins/genetics , Fungal Proteins/genetics , Gene Deletion , Genes, Fungal , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Membrane Proteins/genetics , Molecular Sequence Data , Mutation , Phenotype , Yeasts/geneticsABSTRACT
Vesicular transport to and from the lysosome and late endosome is defective in patients with Chediak-Higashi syndrome (CHS) and in mutant beige (bg) mice. CHS and bg cells have giant, perinuclear vesicles with characteristics of late endosomes and lysosomes that arise from dysregulated homotypic fusion. CHS and bg lysosomes also exhibit compartmental missorting of proteins, such as elastase, glucuronidase and cathepsin G. Lyst, a candidate gene for bg, was identified by direct complementary DNA selection from a yeast artificial chromosome (YAC) clone containing a 650-kilobase segment of the bg-critical region on mouse chromosome 13. Lyst is disrupted by a 5-kilobase deletion in bg mice, and Lyst messenger RNA is markedly reduced in bg homozygotes. The homologous human gene, LYST, is highly conserved with mouse Lyst, and contains a frame-shift mutation at nucleotides 117-118 of the coding domain in a CHS patient. Thus bg mice and human CHS patients have homologous disorders associated with Lyst mutations. Lyst encodes a protein with a carboxy-terminal prenylation motif and multiple potential phosphorylation sites. Lyst protein is predicted to form extended helical domains, and has a region of sequence similar to stathmin, a coiled-coil phosphoprotein thought to act as a relay integrating cellular signal response coupling.