The Golgi is the central organelle of the secretory pathway through which all secretory and plasma membrane
proteins transit and are posttranslationally modified. Our laboratory has three major projects designed to
provide an understanding of the structure function relationship of the Golgi, two projects are global and the
third is focused on identification of the molecules that function in exit from the Golgi in the constitutive
secretory pathway.
The first project is proteome analysis of the endogenous proteins of the Golgi Complex. It is estimated that
approximately 1,000 proteins are resident to this organelle. With the advent of the genome projects, our
ability to identify a protein as novel or known is greatly increased. The Golgi resident proteins from
hepatocytes, small intestine and mammary gland epithelial cells are being mapped using 2D gel electrophoresis
and identified using mass spectrometry. The data obtained will allow major unknown proteins to be identified
and their functions determined. The availability of a Golgi protein database will allow the entire field to take a
more rational approach to studying the multitude of functions that take place within this organelle.
The second project is high resolution 3-D reconstructions of regions of the Golgi ribbon in various cell types
and functional states using high voltage electron microscopy and tomography. This project is carried out in
collaboration with the staff at the laboratory for 3D Fine Structure on the Boulder campus. Structural features
provide the hypothesis for detailed biochemical and molecular experiments.
We have identified six molecules essential for exit from the hepatocyte Golgi
in the constitutive pathway using a cell-free assay that reproduces vesicle
formation from the trans-Golgi. The dimeric TGN38 is the trans-membrane
receptor for the budding complex which consists of p62, a
phosphatidylinositol 3-kinase regulatory subunit, and an associated 25 kD
GTPase, a 100 kD phosphatidylinositol 3-kinase catalytic subunit;
phosphatidylinositol transfer protein and dynamin. Our focus will be to
understand how these molecules interact and identify any missing
components of this transport machinery. Together the data from these
diverse approaches will help us understand the functions of the molecules
required for exit from the Golgi in the constitutive pathway and understand the structural contexts in which
they act.
Selected Publications
Taylor, RS, Jones, SM, Dahl, RH, Nordeen, MH, and Howell, KE (1997)
Characterization of the Golgi complex cleared of protein in transit and examination
of calcium uptake activities. Mol. Biol. Cell 8, 1911-1931.
Taylor, RS, Fialka, I, Jones, SM, Huber, LA, and Howell, KE (1997) Two-dimensional mapping
of the endogenous proeins of the rat hepatocyte Golgi complex cleared of proteins in transit.
Electrophoresis 18, 2601-2612.
Jones SM, and Howell KE (1997)
Phosphatidylinositol 3-kinase is required for formation
of constitutive transport vesicles from the TGN.
J. Cell Biol. 139, 339-349.
Jones SM, Howell KE, henley JR, Cao H,
and McNiven MA (1998) Dynamin participates in the
formation of clathrin-coated and constitutive exocytic
vesicles from the TGN. Science 279,
573-577.
Jones, SM, Alb, JB, Bankaitis, VA, and Howell, KE (1998) Role of Dynamin in the formation of
transport veicles from the trans-Golgi network. Science 279, 573-577.
Jones SM, Alb JG, Jr., Phillips SE,
Bankaitis VA, Howell KE. (1998) A
phosphatidylinositol 3-kinase and phosphatidylinositol
transfer protein act synergistically in formation of
constitutive transport vesicles from the trans-Golgi
network. J. Biol. Chem. 273:10349-10354.
Ladinsky MS, Mastronarde DN, McIntosh
JR, Howell KE, Staehelin LA. (1999) Golgi
structure in three dimensions: Functional insights from
the normal rat kidney cell. J. Cell Biol. 144:1135-1149.
Wu CC, Yates JR 3rd, Neville MC,
Howell KE. (2000) Proteomic analysis of two
functinal states of the Golgi complex in mammary
epithelial cells. Traffic 1:769-782.
Wu CC, Taylor RS, Lane DR, Ladinsky
MS, Weisz, JA, Howell KE. (2000)
GMx33: A novel family of trans-Golgi proteins
identified by proteomics. Traffic 1:963-975.
Wang, J, Howell KE (2000) The
luminal domain of TGN38 interacts with integrin beta 1
and is involved in its trafficking. Traffic
1:713-723.
Marsh JB, Mastronarde DN, Buttle KF,
Howell KE, McIntosh JR. (2001) Organellar
relationships in the Golgi region of the pancreatic beta
cell line, HIT-T15, viaualized by high resolution
electron tomography. Proc. Natl. Acad. Sci. USA
98:2399-2406.
Thompson RJ, Nordeen MH, Howell KE,
Caldwell JH. (2002) A large-conductance
anion channel of the Golgi complex. Biophys J.
83:278-289.
Ladinsky MS, Wu CC, McIntosh S.,
McIntosh JR, Howell KE. (2002) Structure of
the golgi and distribution of reporter molecules at 20
degrees C reveals the complexity of the exit
compartments. Mol. Biol. Cell 13:2810-2825.
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