Kathryn E. Howell

A method for the comprehensive proteomic analysis of membrane proteins.

We describe a method that allows for the concurrent proteomic analysis of both membrane and soluble proteins from complex membrane-containing samples. When coupled with multidimensional protein identification technology (MudPIT), this method results in (i) the identification of soluble and membrane proteins, (ii) the identification of post-translational modification sites on soluble and membrane proteins, and (iii) the characterization of membrane protein topology and relative localization of soluble proteins. Overlapping peptides produced from digestion with the robust nonspecific protease proteinase K facilitates the identification of covalent modifications (phosphorylation and methylation). High-pH treatment disrupts sealed membrane compartments without solubilizing or denaturing the lipid bilayer to allow mapping of the soluble domains of integral membrane proteins. Furthermore, coupling protease protection strategies to this method permits characterization of the relative sidedness of the hydrophilic domains of membrane proteins.

Proteomics of organelles and large cellular structures

The mass-spectrometry-based identification of proteins has created opportunities for the study of organelles, transport intermediates and large subcellular structures. Traditional cell-biology techniques are used to enrich these structures for proteomics analyses, and such analyses provide insights into the biology and functions of these structures. Here, we review the state-of-the-art proteomics techniques for the analysis of subcellular structures and discuss the biological insights that have been derived from such studies.

Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolution electron tomography

The positional relationships among all of the visible organelles in a densely packed region of cytoplasm from an insulin secreting, cultured mammalian cell have been analyzed in three dimensions (3-D) at ≈6 nm resolution. Part of a fast frozen/freeze-substituted HIT-T15 cell that included a large portion of the Golgi ribbon was reconstructed in 3-D by electron tomography. The reconstructed volume (3.1 × 3.2 × 1.2 μm3) allowed sites of interaction between organelles, and between microtubules and organellar membranes, to be accurately defined in 3-D and quantitatively analyzed by spatial density analyses. Our data confirm that the Golgi in an interphase mammalian cell is a single, ribbon-like organelle composed of stacks of flattened cisternae punctuated by openings of various sizes [Rambourg, A., Clermont, Y., & Hermo, L. (1979) Am. J. Anat. 154, 455–476]. The data also show that the endoplasmic reticulum (ER) is a single continuous compartment that forms close contacts with mitochondria, multiple trans Golgi cisternae, and compartments of the endo-lysosomal system. This ER traverses the Golgi ribbon from one side to the other via cisternal openings. Microtubules form close, non-random associations with the cis Golgi, the ER, and endo-lysosomal compartments. Despite the dense packing of organelles in this Golgi region, ≈66% of the reconstructed volume is calculated to represent cytoplasmic matrix. We relate the intimacy of structural associations between organelles in the Golgi region, as quantified by spatial density analyses, to biochemical mechanisms for membrane trafficking and organellar communication in mammalian cells.

Proteomics reveal a link between the endoplasmic reticulum and lipid secretory mechanisms in mammary epithelial cells

The synthesis and secretion of lipids by mammary epithelial cells is a highly ordered process that involves several distinct steps. Triacylglycerols are synthesized in the endoplasmic reticulum and incorporated into microlipid droplets which coalesce into cytoplasmic lipid droplets. These are vectorially transported to the apical plasma membrane where they are secreted into the milk surrounded by a membrane bilayer. The origin of this membrane as well as the mechanism by which cytoplasmic lipid droplets form and become surrounded by membrane is poorly understood. Proteomic analysis of the protein composition of milk fat globules and cytoplasmic lipid droplet has revealed that the endoplasmic reticulum is not only involved in the synthesis of the lipid but also potentially contributes to the membrane component of milk fat globules. The proteins identified suggest possible mechanisms of multiple steps during this process. Completion of the proteome of milk fat globule membranes and cytoplasmic lipid droplets will provide the necessary reporter molecules to follow and dissect the mechanisms of the sorting and ultimate secretion of cytoplasmic lipid droplets.

Predicting function from structure: 3D structure studies of the mammalian golgi complex

3D electron tomography studies of the structure of the mammalian Golgi complex have led to four functional predictions (1). The sorting and exit site from the Golgi comprises two or three distinct trans‐cisternae (2). The docking of vesicular–tubular clusters at the cis‐face and the fragmentation of trans‐cisternae are coordinated (3). The mechanisms of transport through, and exit from, the Golgi vary with physiological state, and in different cells and tissues (4). Specialized trans‐ER functions in the delivery of ceramide to sphingomyelin synthase in the trans‐Golgi membrane, for the regulated sorting via sphingolipid‐cholesterol‐rich domains. These structure‐based predictions can now be tested using a variety of powerful cell and molecular tools.

Two threshold values of low pH block endocytosis at different stages.

The influence of low extracellular pH on endocytosis was studied in baby hamster kidney cells. When the extracellular medium was adjusted to pH 5.7, the intracellular pH decreased within 2 min to pH 6.2 and the endocytosis of horseradish peroxidase (HRP) in the fluid phase dropped to an undetectable level. With an external pH of 6.3, the internal pH dropped to pH 6.8 and HRP was internalized at a normal rate for 5 min but accumulation during longer incubation times did not occur. Morphologically, HRP was visualized in the lumen of a subpopulation of tubular and vesicular endosomes. These observations were confirmed by subcellular fractionation studies using free flow electrophoresis. Low extracellular pH also had an effect on the endocytosis of the membrane‐spanning glycoprotein G of vesicular stomatitis virus which was implanted into the plasma membrane. The internalization of G‐protein was quantitated by a surface fluoroimmunoassay. The endocytosis of G‐protein was not affected when the external pH was dropped to 6.3, but was reduced at an external pH of 5.7. The intracellular ATP was not depleted and the reduction of endocytosis was reversible upon return to physiological pH. Clathrin coated pits were detected by electron microscopy at the plasma membrane of the low‐pH‐treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteomics of rat liver Golgi complex: Minor proteins are identified through sequential fractionation

The discovery of novel proteins resident to the Golgi complex will fuel our future studies of Golgi structure/function and provide justification for proteomic analysis of this organelle. Our approach to Golgi proteomics was to first isolate and characterize the intact organelle free of proteins in transit by use of tissue pretreated with cycloheximide. Then the stacked Golgi fraction was fractionated into biochemically defined subfractions: Triton X‐114 insoluble, aqueous, and detergent phases. The aqueous and detergent phases were further fractionated by anion‐exchange column chromatography. In addition, radiolabeled cytosol was incubated with stacked Golgi fractions containing proteins in transit, and the proteins bound to the Golgi stacks in an energy‐dependent manner were characterized. All fractions were analyzed by two‐dimensional (2‐D) gel electrophoresis and identification numbers were given to 588 unique 2‐D spots. Tandem mass spectrometry was used to analyze 93 of the most abundant 2‐D spots taken from preparative Triton X‐114 insoluble, aqueous and detergent phase 2‐D gels. Fifty‐one known and 22 unknown proteins were identified. This study represents the first installment in the mammalian Golgi proteome database. Our data suggest that cell fractionation followed by biochemical dissection of specific classes of molecules provides a significant advantage for the identification of low abundance proteins in organelles.

A Phosphatidylinositol 3-Kinase and Phosphatidylinositol Transfer Protein Act Synergistically in Formation of Constitutive Transport Vesicles from the Trans-Golgi Network

Current evidence suggests that phosphatidylinositol (PI) kinases and phosphatidylinositol transfer protein (PITP) are involved in driving vesicular traffic from yeast and mammalian trans-Golgi network (TGN). We have tested the interaction between these cytosolic proteins in an assay that measures the formation of constitutive transport vesicles from the TGN in a hepatocyte cell-free system. This reaction is dependent on a novel PI 3-kinase, and we now report that, under conditions of limiting cytosol, purified PI 3-kinase and PITP functionally cooperate to drive exocytic vesicle formation. This synergy was observed with both yeast and mammalian PITPs, and it also extended to the formation of PI 3-phosphate. These collective findings indicate that the PI 3-kinase and PITP synergize to form a pool of PI 3-phosphate that is essential for formation of exocytic vesicles from the hepatocyte TGN.

The mammalian Golgi — complex debates

Since the first description of the Golgi in 1898, key issues regarding this organelle have remained contentious among cell biologists. Resolving these complex debates, which revolve around Golgi structure–function relationships, is prerequisite to understanding how the Golgi fulfils its role as the central organelle and sorting station of the mammalian secretory pathway.

GMx33: A Novel Family of trans-Golgi Proteins Identified by Proteomics

The known functions of the Golgi complex include the sorting, packaging, post‐translational modification, and transport of secretory proteins, membrane proteins, and lipids. Other functions still remain elusive to cell biologists. With the goal of identifying novel Golgi proteins, a proteomics project was undertaken to map the major proteins of the organelle using two‐dimensional gels, to identify the unknowns using tandem mass spectrometry, and to screen for Golgi residents using GFP‐fusion constructs. Multiple unknowns were identified, and the initial characterization of one of these proteins is reported here. GMx33α is a member of a conserved family of cytosolic Golgi‐associated proteins with no known homology to any known functional domain or protein. Biochemical analyses show that GMx33α differentially partitions into all phases of multiple detergent extractions, and two‐dimensional immunoblots reveal that there are multiple differentially modified forms of GMx33α associated with the Golgi, several of which are phosphorylated. Evidence suggests that these post‐translational modifications regulate its association with the Golgi. GMx33α was not found on Golgi budded vesicles, and immuno‐electron microscopy co‐localizes GMx33α to the trans‐face on the same three cisternae as TGN38 in normal rat kidney cells. This work represents the preliminary characterization of a novel family of trans‐Golgi‐associated proteins.