Abstract
The order of enzymatic activity across Golgi cisternae is essential for complex molecule biosynthesis. However, an inability to separate Golgi cisternae has meant that the cisternal distribution of most resident proteins, and their underlying localization mechanisms, are unknown. Here, we exploit differences in surface charge of intact cisternae to perform separation of early to late Golgi subcompartments. We determine protein and glycan abundance profiles across the Golgi; over 390 resident proteins are identified, including 136 new additions, with over 180 cisternal assignments. These assignments provide a means to better understand the functional roles of Golgi proteins and how they operate sequentially. Protein and glycan distributions are validated in vivo using high-resolution microscopy. Results reveal distinct functional compartmentalization among resident Golgi proteins. Analysis of transmembrane proteins shows several sequence-based characteristics relating to pI, hydrophobicity, Ser abundance, and Phe bilayer asymmetry that change across the Golgi. Overall, our results suggest that a continuum of transmembrane features, rather than discrete rules, guide proteins to earlier or later locations within the Golgi stack.
Highlights
The Golgi is an ancient organelle, common to all eukaryotic lineages (Klute et al, 2011), consisting of a stack of flattened, membranous discs, or cisternae, in which protein and lipid cargoes are modified in a progressive manner and substituted with complex glycan side chains (Ito et al, 2014; Strasser, 2016; van de Meene et al, 2017)
Nonmembrane components from the samples peaked in early fractions outside this range. (C) Endomembrane fractions were primarily investigated using shotgun proteomics to measure the relative amounts of the different proteins contained therein
Proteins were identified via the mass fingerprints of trypic digest peptides searched against the most recent Arabidopsis proteome using MASCOT software. (D) Average Free-flow electrophoresis (FFE) abundance profiles for resident proteins from Golgi, endoplasmic reticulum (ER), and other organelles using independent subcellular localizations derived from localization of organelle proteins by isotope tagging (LOPIT) analysis (Supplemental Data Set 1)
Summary
The Golgi is an ancient organelle, common to all eukaryotic lineages (Klute et al, 2011), consisting of a stack of flattened, membranous discs, or cisternae, in which protein and lipid cargoes are modified in a progressive manner and substituted with complex glycan side chains (Ito et al, 2014; Strasser, 2016; van de Meene et al, 2017). The Golgi is the hub of the secretory pathway, trafficking cargo-containing vesicles to and from the endoplasmic reticulum (ER) at the cis face (Brandizzi and Barlowe, 2013) and to other cellular destinations at the trans face (Gendre et al, 2015). There have been important advances in understanding trafficking processes from the trans-Golgi network (TGN) to post-TGN. Studying secretory organelle organization contributes to a general understanding of biochemical pathways and how protein localization is specified and gives us the capacity to better control the complex, sequential biochemistry and trafficking processes of cellular secretion. Understanding of how sequence characteristics localize proteins to organelles has advanced (Sharpe et al, 2010), no general sequence-based determinants of Golgi cisternal membrane localization are known (Banfield, 2011). Too few proteins have been accurately localized within the Golgi to identify cisternal targeting sequences or map intra-Golgi trafficking pathways
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