In 1898, Camillo Golgi described the diffuse reticular network that bears his name.12
A half-century later, the ribbonlike cisternal architecture of this intracellular structure was observed with an electron microscope.13
How the Golgi apparatus forms, how it maintains its architecture, and what the relationship is between its structure and its functions — which include glycosylation of proteins — remain incompletely understood.14
Golgin proteins participate in these processes; ex vivo knockdown of the golgins GMAP-210, golgin-84, and GM130 result in fragmentation of the Golgi apparatus.15-17
Golgi fragmentation does not completely block the transport and processing of nascent proteins in cultured cells, but it does reduce the efficiency of these processes.15,16
The identification of mice with a loss-of-function mutation in the gene encoding GMAP-210 facilitates assessment of the function of the Golgi structure in vivo. Although additional roles of GMAP-210 have been suggested,7,18-20
our in vivo data are most consistent with its role in Golgi organization and protein processing.
Despite expression of GMAP-210 in many cell types, GMAP-210 deficiency altered Golgi architecture and caused swelling of the endoplasmic reticulum in a subgroup of tissues. Cells within the developing skeletal system — chondrocytes and osteoblasts — have very high rates of matrix secretion21
and may be particularly susceptible to stress when Golgi processing is inefficient. Indeed, chondrocytes with mutant GMAP-210 showed clear evidence of stress in the endoplasmic reticulum, abnormal differentiation, and increased cell death. Impaired protein processing and swelling of the endoplasmic reticulum have been observed in other human skeletal dysplasias. Typically, these dysplasias have been caused by mutations in a secreted matrix protein that impair its folding and secretion. Examples include specific mutations of type I collagen in osteogenesis imperfecta,22
type II collagen in spondyloepiphyseal dysplasia congenita,23
type X collagen in Schmid metaphyseal dysplasia,24
and cartilage oligomeric matrix protein in pseudoachondroplasia and multiple epiphyseal dysplasia.25
Investigators have identified mutations in patients with craniolenticular dysplasia that affect secretory mutant protein 23A (SEC23A), a protein involved in generating vesicles containing coat protein complex II that are used to move proteins from the endoplasmic reticulum to the Golgi apparatus.26
The nonlethality of SEC23A deficiency may be explained by the coexpression of its paralog, SEC23B, in most cell types.27,28
Mutations affecting trafficking protein particle complex 2 (TRAPPC2) and dymeclin, two other proteins thought to have roles in the transport of proteins between the endoplasmic reticulum and the Golgi apparatus, also cause nonlethal skeletal dysplasias — X-linked spondyloepiphyseal dysplasia tarda and the Dyggve–Melchior–Clausen syndrome, respectively.29-31
Therefore, it is reasonable to postulate that the developing skeleton, being sensitive to defects in protein transport, will be affected by mutations in other components of the secretory pathways whose deficiency causes Golgi disorganization.
Important post-translational modifications, such as the synthesis of glycosaminoglycans, occur in the Golgi apparatus. Chondrocytes produce several highly modified proteoglycans, the most abundant of which is aggrecan, a very large proteoglycan (measuring 500 nm and having a mass of 3 megadaltons) that contains hundreds of glycosaminoglycan side chains.32
Immunofluorescence studies showed that aggrecan core protein was present in both wild-type and mutant cartilage, even though staining with Alcian blue, electron microscopy, and lectin staining suggest that this protein is improperly glycosylated in the mutant cartilage. In contrast, perlecan, a less abundant and smaller glycosaminoglycan containing proteoglycan, is abnormally retained within mutant chondrocytes and fibroblasts. These data suggest that GMAP-210 maintains the Golgi structure in chondrocytes, thereby facilitating complex glycosylation reactions while also participating in the transport of specific proteins. Indeed, RNAi knockdown of golgin-97 and golgin-245 has been shown to affect the secretion of E-cadherin and tumor necrosis factor α
, respectively. However, depletion of these golgins did not affect the organization of the Golgi apparatus.33,34
Loss-of-function mutations in solute carrier family 26, member 2 (SLC26A2
), which encodes a high-affinity sulfate transporter, cause achondrogenesis type 1B, which is phenotypically similar to achondrogenesis type 1A.35
Impaired post-translational modification of proteoglycans in cartilage appears to be common to the two disorders.
Follit and colleagues described neonatal lethality in mice with a gene-trap insertion into Trip11
Their studies focused on the role of GMAP-210 in the formation of the primary cilia and in intraflagellar transport; they noted that cells from the mutant mice had shorter cilial than those from wild-type mice and that these cells failed to localize intraflagellar transport protein 20 to the Golgi apparatus. Impaired cilial function has previously been shown to cause skeletal-patterning defects as well as skeletal dysplasia through disruption of hedgehog signaling.36,37
Our data suggest that neither impaired cilial function nor impaired hedgehog signaling is a primary contributor to the skeletal phenotype in mice lacking GMAP-210 (Fig. 7 in the Supplementary Appendix
). Decreased alveolar formation was observed in the lungs of gene-trap mutant mice,7
which we also observed in our mutant mice. We suggest that the lung phenotype is a consequence of the skeletal phenotype, since alveolar deficiency has been observed in other conditions characterized by insufficient thoracic volume.38
Although we found TRIP11
mutations in all 10 patients with achondrogenesis type 1A that were tested, we cannot conclude that every case is attributable to mutations in TRIP11
. However, the identification of TRIP11
as the major disease-causing gene makes it possible to conduct genetic testing in families with a history of achondrogenesis type 1A. It is notable that we did not observe missense mutations in patients with achondrogenesis type 1A, suggesting that milder mutations may result in a different clinical phenotype. For example, SNPs near TRIP11
have been associated with normal variation in human height.39,40
Forward genetic analysis has the potential to provide insight into the molecular mechanisms of a biologic process that is novel and unexpected, since a phenotype-driven screen is not biased by preconceptions regarding gene function. In vitro knockdown of GMAP-210 had suggested that the absence of this protein would lead to Golgi fragmentation and dysfunction in all cells. Instead, a forward genetic screen in mutagenized mice indicated that the absence of GMAP-210 predominantly affects the development of the skeletal system. Our finding that patients with achondrogenesis type 1A, a phenotypically similar syndrome, also have mutations in TRIP11 that lead to GMAP-210 deficiency underscores the power and usefulness of this strategy of genetic inquiry.