Fergus J. Doherty

Ubiquitinated protein conjugates are specifically enriched in the lysosomal system of fibroblasts

Ubiquitin‐protein conjugates are found by imniunogold electron microscopy to be enriched (12‐fold) in the lysosomal compartment of 3T3‐L1 fibroblasts. Treatment of fibroblasts with the cysteine protease inhibitor E‐64 leads to an expansion of the lysosomal compartment and as a result an increase in the cellular content of ubiquitin‐protein conjugates. There is no change in the specific enrichment of ubiquitin‐protein conjugates in the lysosomal compartment following E‐64 treatment. The results suggest that some ubiquitin‐protein conjugates may normally be degraded lysosomally following sequestration by microautophagy and imply that protein ubiquitination may be one of the signals for protein uptake into lysosomes.

Intracellular protein catabolism: state of the art

There are several reasons why intracellular protein catabolism is incompletely understood. The most quoted are the facts that protein catabolism involves multiple mechanisms and does not easily occur in the test tube, at least, to a readily manipulatable degree (cf. protein synthesis). However, a much more fundamental reason is that the multiple systems are intimately connected with all aspects of the life process. Degradation is the alternative for proteins during or immediately after synthesis, during secretion and after reaching their cytomorphological functional sites: in short, protein catabolism (including degradation after protein endocytosis) is primarily a cell biological phenomenon. Extracting the molecular detail of the multiple processes requires multiple biological approaches, providing a literature rich in a mixture of experimental models, designs and interpretations. In this review we want to consider critically the biological questions, the literature which addresses the questions and the current conundrums: the state of the art. We have taken the excellent 1982 review of Hershko and Ciechanover [l] as representative of knowledge and understanding in 1981 and have predominantly reviewed the literature from 1981 to 85. We have not considered degradative events after endocytosis since they are adequately addressed elsewhere [2]. Those people interested in the views of many workers in the field would do well to read the summaries of presentations at the Vth International Symposium on Protein Catabolism [3]. Hershko’s analyses [l] saw intracellular protein catabolism as an extensive, selective set of energy(ATP)-requiring processes which have several functions including elimination of abnormal proteins and provision of amino acids in times of need. Non-lysosomal and lysosomal systems were identified which may act on ‘short’ and ‘long’ lived proteins respectively: the latter system being activated in a variety of cellular deprivation states. Finally and importantly, much of the earlier experimentation was seen as phenomenological rather than molecular in nature. The analyses did not consider: (i) the question of cellular architecture, i.e. proteins function in defined sites and yet must interact with the catabolic systems; (ii) that proteins are really degraded with the rate of heterogeneity previously supposed; (iii) the existence of ‘special’ systems, e.g. in heat shock or regulatory proteases responsive to calcium; (iv) degradation of proteins during secretion; (v) mitochondrial and chloroplast systems. We will consider intracellular protein catabolism from the point of view of (section 2) the systems: (section 2.1) the lysosomal system, (2.2) the secretory degradative system (defined as secretioncoupled degradation), (2.3) non-lysosomal systems, (2.4) ATP-dependent systems in prokaryotes, eukaryotes and energy-transducing organelles, (2.5) calcium-dependent systems; and (section 3) the substrates: (3.1) how are the systems organised topographically with respect to

Inclusion bodies in motor cortex and brainstem of patients with motor neurone disease are detected by immunocytochemical localisation of ubiquitin.

Histological sections of cerebral motor cortex, brainstem, and spinal cord from 10 cases of clinically diagnosed motor neurone disease (MND) and 10 control cases were examined by conventional histology and immunocytochemical methods to localise ubiquitin. Intracytoplasmic inclusion bodies were identified in motor neurones of hypoglossal nuclei and appeared specific for MND. Similar inclusions were found in both large pyramidal cells and small neurones in the motor cortex, and were restricted to 4 cases having the amyotrophic lateral sclerosis form of MND with severe degeneration of corticospinal tracts. As reported in earlier studies, cellular inclusion bodies were identified in motor neurones of spinal cord from cases of MND but not in control material. Ubiquitin inclusions in motor neurones appear to be markers for the degenerative process causing neuronal loss in MND and there appears to be a close association between the anatomical location of inclusions and clinical manifestations of disease.

Immunogold localisation of ubiquitin‐protein conjugates in primary (azurophilic) granules of polymorphonuclear neutrophils

Ubiquitin—protein conjugates are found in the primary (azurophilic) lysome‐related granules but not in the secondary (specific) granules in mature polymorphonuclear neutrophils prepared from bone marrow. This is the first reported demonstration of ubiquitin—protein conjugates in lysosome‐related membrane‐bound vesicles in granulocytes and complements our previous findings of ubiquitinated proteins in lysosomes of fibroblasts. The significance of the selective presence of conjugates in only one of the two main types of neutrophil granules remains to be elucidated but may relate to the presence of the complement of acid hydrolases, including proteases, in the azurophilic granules compared to the specific granules. Ubiquitin—protein conjugates may enter the primary granules during neutrophil maturation by an autophagic process or by a heterophagic process during the fusion of phagosomes with primary granules. Alternatively protein ubiquitination may be involved in granule biogenesis.

Immunogold localisation of ubiquitin-protein conjugates in Sf9 insect cells. Implications for the biogenesis of lysosome-related organelles

Immunogold electron microscopy with antibodies which primarily detect ubiquitin‐protein conjugates shows conjugate‐specific gold particles enriched severalfold in acid phosphatase‐positive lysosomes and multivesicular bodies in insect Sf9 cells. The observations demonstrate that ubiquitinated proteins are associated with small acid phosphatase‐containing primary lysosomes (transport vesicles) and indicate a pathway in which primary lysosomes fuse with multivesicular bodies to generate mature lysosome‐related structures.

RELATED ORGANELLES OF THE ENDOSOME-LYSOSOME SYSTEM CONTAIN A DIFFERENT REPERTOIRE OF UBIQUITINATED PROTEINS IN SF9 INSECT CELLS

Two components of the endosomal/lysosomal compartment of Sf9 cells, multivesicular bodies (MVB) and light vacuoles with membrane complexes (LVMC) have been isolated and probed for ubiquitin protein conjugates with a specific antibody. Immunogold electron microscopy indicates that whereas ubiquitin‐protein conjugates are localised to electron dense areas of MVB they are associated with the membranes of LVMC. Five ubiquitinated polypeptides are revealed in MVB by immunoblotting while numerous ubiquitinated species forming a smear following electrophoresis are present in LVMC. We suggest two possible routes for entry of ubiquitin‐protein conjugates into these organelles, via the cell surface and via primary lysosomes.

Insoluble disulfide cross-linked polypeptides accumulate in the functionally compromised lysosomes of fibroblasts treated with the cysteine protease inhibitor E-64.

Mouse fibroblasts (3T3-L1 cells) accumulate pulse-labeled long-lived polypeptides in detergent- and salt-insoluble aggregates when chased in the presence of inhibitors of lysosomal cysteine cathepsins, including E-64. Proteins found in the detergent- and salt-insoluble fraction include polypeptides which are disulfide cross-linked. E-64-induced polypeptide aggregates cofractionate with lysosomal enzyme markers on density gradients and are found in multivesicular dense bodies which by electron microscopy appear to be engaged in microautophagy. The results are discussed in relation to the possible role of polypeptide aggregation in the sequestration or trapping of cytoplasmic proteins by the lysosomal system.